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Abstract:

Disclosed are mutant DNA polymerases having increased 3'-mismatch
discrimination relative to a corresponding, unmodified polymerase. The
mutant polymerases are useful in a variety of disclosed primer extension
methods. Also disclosed are related compositions, including recombinant
nucleic acids, vectors, and host cells, which are useful, e.g., for
production of the mutant DNA polymerases.

10. The DNA polymerase of claim 3, wherein the polymerase has at least
80% sequence identity to SEQ ID NO:1.

11. The DNA polymerase of claim 10, wherein the DNA polymerase is a Z05
DNA polymerase, and the amino acid at position 580 of SEQ ID NO:1 is
selected from the group consisting of L, G, T, Q, A, S, N, R, and K.

13. A method for conducting primer extension, comprising: contacting a
DNA polymerase according to claim 1 with a primer, a polynucleotide
template, and nucleoside triphosphates under conditions suitable for
extension of the primer, thereby producing an extended primer.

14. The method of claim 13, wherein the primer extension method is a
method for conducting polymerase chain reaction (PCR).

15. A kit for producing an extended primer, comprising: at least one
container providing a DNA polymerase according to claim 1.

16. The kit according to claim 15, further comprising one or more
additional containers selected from the group consisting of: (a) a
container providing a primer hybridizable, under primer extension
conditions, to a predetermined polynucleotide template; (b) a container
providing nucleoside triphosphates; and (c) a container providing a
buffer suitable for primer extension.

17. A reaction mixture comprising a DNA polymerase according to claim 1,
at least one primer, a polynucleotide template, and nucleoside
triphosphates.

Description:

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present invention claims benefit of priority to U.S.
Provisional Patent Application No. 61/356,279, filed Jun. 18, 2010, which
is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention provides DNA polymerases with increased
3'-mismatch discrimination and their use in various applications,
including nucleic acid polynucleotide extension and amplification.

BACKGROUND OF THE INVENTION

[0003] DNA polymerases are responsible for the replication and maintenance
of the genome, a role that is central to accurately transmitting genetic
information from generation to generation. DNA polymerases function in
cells as the enzymes responsible for the synthesis of DNA. They
polymerize deoxyribonucleoside triphosphates in the presence of a metal
activator, such as Mg2+, in an order dictated by the DNA template or
polynucleotide template that is copied. In vivo, DNA polymerases
participate in a spectrum of DNA synthetic processes including DNA
replication, DNA repair, recombination, and gene amplification. During
each DNA synthetic process, the DNA template is copied once or at most a
few times to produce identical replicas. In contrast, in vitro, DNA
replication can be repeated many times such as, for example, during
polymerase chain reaction (see, e.g., U.S. Pat. No. 4,683,202).

[0004] In the initial studies with polymerase chain reaction (PCR), the
DNA polymerase was added at the start of each round of DNA replication
(see U.S. Pat. No. 4,683,202, supra). Subsequently, it was determined
that thermostable DNA polymerases could be obtained from bacteria that
grow at elevated temperatures, and that these enzymes need to be added
only once (see U.S. Pat. No. 4,889,818 to Gelfand and U.S. Pat. No.
4,965,188 to Mullis). At the elevated temperatures used during PCR, these
enzymes are not irreversibly inactivated. As a result, one can carry out
repetitive cycles of polymerase chain reactions without adding fresh
enzymes at the start of each synthetic addition process. DNA polymerases,
particularly thermostable polymerases, are the key to a large number of
techniques in recombinant DNA studies and in medical diagnosis of
disease. For diagnostic applications in particular, a target nucleic acid
sequence may be only a small portion of the DNA or RNA in question, so it
may be difficult to detect the presence of a target nucleic acid sequence
without amplification.

[0005] The overall folding pattern of DNA polymerases resembles the human
right hand and contains three distinct subdomains of palm, fingers, and
thumb. (See Beese et al., Science 260:352-355, 1993); Patel et al.,
Biochemistry 34:5351-5363, 1995). While the structure of the fingers and
thumb subdomains vary greatly between polymerases that differ in size and
in cellular functions, the catalytic palm subdomains are all
superimposable. For example, motif A, which interacts with the incoming
dNTP and stabilizes the transition state during chemical catalysis, is
superimposable with a mean deviation of about one Å amongst mammalian
pol α and prokaryotic pol I family DNA polymerases (Wang et al.,
Cell 89:1087-1099, 1997). Motif A begins structurally at an antiparallel
β-strand containing predominantly hydrophobic residues and continues
to an α-helix. The primary amino acid sequence of DNA polymerase
active sites is exceptionally conserved. In the case of motif A, for
example, the sequence DYSQIELR (SEQ ID NO:28) is retained in polymerases
from organisms separated by many millions years of evolution, including,
e.g., Thermus aquaticus, Chlamydia trachomatis, and Escherichia coli.

[0006] In addition to being well-conserved, the active site of DNA
polymerases has also been shown to be relatively mutable, capable of
accommodating certain amino acid substitutions without reducing DNA
polymerase activity significantly. (See, e.g., U.S. Pat. No. 6,602,695)
Such mutant DNA polymerases can offer various selective advantages in,
e.g., diagnostic and research applications comprising nucleic acid
synthesis reactions. Thus, there is a need in the art for identification
of amino acid positions amenable to mutation to yield improved polymerase
activities. The present invention, as set forth herein, meets these and
other needs.

BRIEF SUMMARY OF THE INVENTION

[0007] Provided herein are DNA polymerases having increased 3'-mismatch
discrimination relative to a corresponding, unmodified control
polymerase, and methods of making and using such DNA polymerases. In some
embodiments, the polymerase is a thermostable DNA polymerase. In some
embodiments, the DNA polymerase is a thermoactive DNA polymerase. In some
embodiments, the DNA polymerase of the present invention is derived from
a Thermus species. In some embodiments, the DNA polymerase is derived
from a Thermotoga species.

[0008] As described herein, the inventors discovered that the amino acid
of the DNA polymerase corresponding to position 493 of SEQ ID NO:1 can be
mutated from the native amino acid at that position to produce an enzyme
having increased 3'-mismatch discrimination relative to a corresponding,
unmodified control polymerase. In DNA polymerases derived from a Thermus
species, the native amino acid corresponding to position 493 of SEQ ID
NO:1 is E. In DNA polymerases derived from a Thermotoga species, the
native amino acid corresponding to position 493 of SEQ ID NO:1 is S. In
DNA polymerases derived from other non-Thermus, non-Thermotoga species,
the amino acid corresponding to position 493 of SEQ ID NO:1 can be an
amino acid other than E or S, for example, A or G. See, FIG. 1.

[0009] In some embodiments, the amino acid of the DNA polymerase
corresponding to position 493 of SEQ ID NO:1 is any amino acid other than
E, S, A or G, and the control DNA polymerase has the same amino acid
sequence as the DNA polymerase except that the amino acid of the control
DNA polymerase corresponding to position 493 of SEQ ID NO:1 is E, S, A or
G. For example, in some embodiments, the amino acid at the position
corresponding to position 493 of SEQ ID NO:1 is selected from V, L, I, M,
F, W, P, T, C, Y, N, Q, D, K, R or H.

[0010] However, the inventors have further discovered that insertion of A
or G (the native amino acid in some non-Thermus, non-Thermotoga species)
in place of E at position 493 of Thermus polymerases also increased
3'-mismatch discrimination. Thus, in some embodiments, the DNA polymerase
of the invention is derived from a Thermus species, and the amino acid of
the DNA polymerase corresponding to position 493 of SEQ ID NO:1 is any
amino acid other than E, and the control DNA polymerase has the same
amino acid sequence as the DNA polymerase except that the amino acid of
the control DNA polymerase corresponding to position 493 of SEQ ID NO:1
is E. For example, where the DNA polymerase is derived from a Thermus
species, the amino acid at the position corresponding to position 493 of
SEQ ID NO:1 can be selected from S, A, G, V, L, I, M, F, W, P, T, C, Y,
N, Q, D, K, R or H. In some embodiments the amino acid at the position
corresponding to position 493 of SEQ ID NO:1 is selected from S, A, Q, G,
K, or R. In some embodiments the amino acid at the position corresponding
to position 493 of SEQ ID NO:1 is selected from A, G, K, or R. In some
embodiments the amino acid at the position corresponding to position 493
of SEQ ID NO:1 is K.

[0011] In some embodiments, the DNA polymerase of the invention is derived
from a Thermus species, and the amino acid corresponding to position 493
of SEQ ID NO:1 is an amino acid having a polar, uncharged side-chain
(e.g., N, Q, H, S, T, or Y), a nonpolar, uncharged side-chain (e.g., G,
A, L, M, W, P, F, C, V, or I), or a polar, positively charged side-chain
(e.g., R or K) at neutral pH. In some embodiments, the amino acid
corresponding to position 493 of SEQ ID NO:1 having a nonpolar, uncharged
side-chain is A or G. In some embodiments, the amino acid corresponding
to position 493 of SEQ ID NO:1 having a polar, positively charged
side-chain is R or K. In some embodiments, the amino acid corresponding
to position 493 of SEQ ID NO:1 having a polar, positively charged
side-chain is K.

[0012] Further, the inventors found that other amino acids, located nearby
to the amino acid corresponding to position 493 of SEQ ID NO:1, can also
be mutated to produce an enzyme having increased 3'-mismatch
discrimination relative to a corresponding, unmodified control
polymerase. For example, mutations at amino acids corresponding to
positions 488, and/or 497 of SEQ ID NO:1 also produce an enzyme having
increased 3'-mismatch discrimination relative to a corresponding,
unmodified control polymerase.

[0018] In some embodiments, the DNA polymerase having improved 3'-mismatch
discrimination comprises a motif in the polymerase domain comprising
[0019] A-G-H-P-F-N-L-N-S-R-D-Q-L-X10-R-V-L-F-D-E-L, wherein: [0020]
X10 is any amino acid other than E (SEQ ID NO:9).

[0027] In some embodiments, the amino acid of the DNA polymerase
corresponding to position 580 of SEQ ID NO:1 is any amino acid other than
D or E. In some embodiments, the amino acid of the DNA polymerase
corresponding to position 580 of SEQ ID NO:1 is any amino acid other than
D. In some embodiments, the amino acid of the DNA polymerase
corresponding to position 580 of SEQ ID NO:1 is selected from the group
consisting of L, G, T, Q, A, S, N, R and K. In some embodiments, the
amino acid of the DNA polymerase corresponding to position 580 of SEQ ID
NO:1 is G.

[0028] In some embodiments, the DNA polymerase further comprises a
mutation at one or more amino acids corresponding to a position selected
from 488 and/or 497 SEQ ID NO:1. In some embodiments, the amino acid of
the DNA polymerase corresponding to position 488 of SEQ ID NO:1 is any
amino acid other than S. In some embodiments, the amino acid of the DNA
polymerase corresponding to position 488 of SEQ ID NO:1 is selected from
G, A, V, L, I, M, F, W, P, E, T, C, N, Q, D, Y, K, R or H. In some
embodiments, the amino acid of the DNA polymerase corresponding to
position 488 of SEQ ID NO:1 is G, A, D, F, K, C, T, or Y. In some
embodiments, the amino acid of the DNA polymerase corresponding to
position 497 of SEQ ID NO:1 is any amino acid other than F or Y. In some
embodiments, the amino acid of the DNA polymerase corresponding to
position 497 of SEQ ID NO:1 is selected from R, V, L, I, M, W, P, T, C,
N, D, E, S, A, G, K, E or H. In some embodiments, the DNA polymerase is
derived from a Thermus species, and the amino acid of the DNA polymerase
corresponding to position 497 of SEQ ID NO:1 is any amino acid other than
F. Thus, in some embodiments, the amino acid of the DNA polymerase
corresponding to position 497 of SEQ ID NO:1 is A, G, S, T, Y, D, or K.

[0042] In some embodiments, the DNA polymerase has at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% sequence identity to SEQ ID NO:1. In some embodiments, the
DNA polymerase is a Thermus sp. Z05 DNA polymerase (Z05) DNA polymerase
(i.e., SEQ ID NO:1), except that the amino acid at position 493 is any
amino acid other than E or S. In some embodiments, the DNA polymerase is
a Z05 DNA polymerase, and the amino acid at position 493 is any amino
acid other than E. For example, in some embodiments, the amino acid at
position 493 is selected from V, L, I, M, F, W, P, T, C, Y, N, Q, D, K,
R, S, A, G or H. In some embodiments, the DNA polymerase is a Z05 DNA
polymerase, and the amino acid at position 493 is G, A, K, or R. In some
embodiments, the DNA polymerase is a Z05 DNA polymerase, and the amino
acid at position 493 is K. In some embodiments, the DNA polymerase is a
Z05 DNA polymerase further comprising a substitution at position 580, and
the amino acid at position 580 is any amino acid other than D or E. In
some embodiments, the DNA polymerase is a Z05 DNA polymerase, and the
amino acid at position 580 is any amino acid other than D. In some
embodiments, the DNA polymerase is a Z05 DNA polymerase, and the amino
acid at position 580 is selected from the group consisting of L, G, T, Q,
A, S, N, R and K. In some embodiments, the DNA polymerase is a Z05 DNA
polymerase, and the amino acid at position 580 is G.

[0043] The mutant or improved polymerase can include other,
non-substitutional modifications. One such modification is a thermally
reversible covalent modification that inactivates the enzyme, but which
is reversed to activate the enzyme upon incubation at an elevated
temperature, such as a temperature typically used for polynucleotide
extension. Exemplary reagents for such thermally reversible modifications
are described in U.S. Pat. Nos. 5,773, 258 and 5,677,152 to Birch et al.,
which are expressly incorporated by reference herein in their entirety.

[0044] In some embodiments, the 3'-mismatch activity is determined using a
mutant BRAF V600R target polynucleotide having the nucleic acid sequence
of SEQ ID NO:35 (wild type BRAF=SEQ ID NO:34) in the presence of a
forward primer that is perfectly matched to the mutant sequence and has a
single 3' A:C mismatch to the wild type sequence in one or more reaction
mixtures having a predetermined number of copies of wild-type BRAF V600
target polynucleotide and a predetermined number of copies of mutant BRAF
V600R target polynucleotide equal in number or fewer than the number of
copies of wild-type target (e.g., 10,000 or fewer copies). Two or more
reaction mixtures can have titrated numbers of copies of mutant BRAF
V600R target polynucleotide (e.g., 1:5 titrations, 1:10 titrations, e.g.,
10,000 copies, 1000 copies, 100 copies, 10 copies, 1 copy, 0 copies in
several reaction mixtures). The 3'-mismatch discrimination ability of a
polymerase of the invention can be compared to the 3'-mismatch
discrimination ability of a reference polymerase (e.g., a naturally
occurring or unmodified polymerase), over a preselected unit of time, as
described herein. Polymerases with increased 3'-mismatch discrimination
ability will not amplify the wild-type sequence when contacted with a
primer that is perfectly matched to a mutant allele, or will require a
greater number of PCR cycles to amplify the wild-type sequence using the
mutant allele-specific primer (i.e., exhibit a higher Cp value), in
comparison to a naturally occurring or unmodified polymerase.

[0045] In various other aspects, the present invention provides a
recombinant nucleic acid encoding a mutant or improved DNA polymerase as
described herein, a vector comprising the recombinant nucleic acid,
and/or a host cell transformed with the vector. In certain embodiments,
the vector is an expression vector. Host cells comprising such expression
vectors are useful in methods of the invention for producing the mutant
or improved polymerase by culturing the host cells under conditions
suitable for expression of the recombinant nucleic acid. The polymerases
of the invention may be contained in reaction mixtures and/or kits. The
embodiments of the recombinant nucleic acids, host cells, vectors,
expression vectors, reaction mixtures and kits are as described above and
herein.

[0046] In yet another aspect, a method for conducting polynucleotide
extension is provided. The method generally includes contacting a DNA
polymerase having increased 3'-mismatch discrimination as described
herein with a primer, a polynucleotide template, and nucleoside
triphosphates under conditions suitable for extension of the primer,
thereby producing an extended primer. The polynucleotide template can be,
for example, an RNA or DNA template. The nucleoside triphosphates can
include unconventional nucleotides such as, e.g., ribonucleotides and/or
labeled nucleotides. Further, the primer and/or template can include one
or more nucleotide analogs. In some variations, the polynucleotide
extension method is a method for polynucleotide amplification that
includes contacting the mutant or improved DNA polymerase with a primer
pair, the polynucleotide template, and the nucleoside triphosphates under
conditions suitable for amplification of the polynucleotide. The
polynucleotide extension reaction can be, e.g., PCR, isothermal
extension, or sequencing (e.g., 454 sequencing reaction).

[0047] The present invention also provides a kit useful in such a
polynucleotide extension method. Generally, the kit includes at least one
container providing a mutant or improved DNA polymerase as described
herein. In certain embodiments, the kit further includes one or more
additional containers providing one or more additional reagents. For
example, in specific variations, the one or more additional containers
provide nucleoside triphosphates; a buffer suitable for polynucleotide
extension; and/or a primer hybridizable, under polynucleotide extension
conditions, to a predetermined polynucleotide template.

[0050] Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention pertains. Although essentially
any methods and materials similar to those described herein can be used
in the practice or testing of the present invention, only exemplary
methods and materials are described. For purposes of the present
invention, the following terms are defined below.

[0054] The term "aptamer" refers to a single-stranded DNA that recognizes
and binds to DNA polymerase, and efficiently inhibits the polymerase
activity as described in U.S. Pat. No. 5,693,502, hereby expressly
incorporated by reference herein in its entirety.

[0055] The term "mutant," in the context of DNA polymerases of the present
invention, means a polypeptide, typically recombinant, that comprises one
or more amino acid substitutions relative to a corresponding,
naturally-occurring or unmodified DNA polymerase.

[0056] The term "unmodified form," in the context of a mutant polymerase,
is a term used herein for purposes of defining a mutant DNA polymerase of
the present invention: the term "unmodified form" refers to a functional
DNA polymerase that has the amino acid sequence of the mutant polymerase
except at one or more amino acid position(s) specified as characterizing
the mutant polymerase. Thus, reference to a mutant DNA polymerase in
terms of (a) its unmodified form and (b) one or more specified amino acid
substitutions means that, with the exception of the specified amino acid
substitution(s), the mutant polymerase otherwise has an amino acid
sequence identical to the unmodified form in the specified motif The
"unmodified polymerase" (and therefore also the modified form having
increased 3'-mismatch discrimination) may contain additional mutations to
provide desired functionality, e.g., improved incorporation of
dideoxyribonucleotides, ribonucleotides, ribonucleotide analogs,
dye-labeled nucleotides, modulating 5'-nuclease activity, modulating
3'-nuclease (or proofreading) activity, or the like. Accordingly, in
carrying out the present invention as described herein, the unmodified
form of a DNA polymerase is predetermined. The unmodified form of a DNA
polymerase can be, for example, a wild-type and/or a naturally occurring
DNA polymerase, or a DNA polymerase that has already been intentionally
modified. An unmodified faun of the polymerase is preferably a
thermostable DNA polymerases, such as DNA polymerases from various
thermophilic bacteria, as well as functional variants thereof having
substantial sequence identity to a wild-type or naturally occurring
thermostable polymerase. Such variants can include, for example, chimeric
DNA polymerases such as, for example, the chimeric DNA polymerases
described in U.S. Pat. No. 6,228,628 and U.S. Application Publication No.
2004/0005599, which are incorporated by reference herein in their
entirety. In certain embodiments, the unmodified form of a polymerase has
reverse transcriptase (RT) activity.

[0057] The term "thermostable polymerase," refers to an enzyme that is
stable to heat, is heat resistant, and retains sufficient activity to
effect subsequent polynucleotide extension reactions and does not become
irreversibly denatured (inactivated) when subjected to the elevated
temperatures for the time necessary to effect denaturation of
double-stranded nucleic acids. The heating conditions necessary for
nucleic acid denaturation are well known in the art and are exemplified
in, e.g., U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,965,188, which are
incorporated herein by reference. As used herein, a thermostable
polymerase is suitable for use in a temperature cycling reaction such as
the polymerase chain reaction ("PCR"). Irreversible denaturation for
purposes herein refers to permanent and complete loss of enzymatic
activity. For a thermostable polymerase, enzymatic activity refers to the
catalysis of the combination of the nucleotides in the proper manner to
form polynucleotide extension products that are complementary to a
template nucleic acid strand. Thermostable DNA polymerases from
thermophilic bacteria include, e.g., DNA polymerases from Thermotoga
maritima, Thermus aquaticus, Thermus thermophilus, Thermus flavus,
Thermus filiformis, Thermus species Sps17, Thermus species Z05, Thermus
caldophilus, Bacillus caldotenax, Thermotoga neopolitana, and Thermosipho
africanus.

[0058] The term "thermoactive" refers to an enzyme that maintains
catalytic properties at temperatures commonly used for reverse
transcription or anneal/extension steps in RT-PCR and/or PCR reactions
(i.e., 45-80° C.). Thermostable enzymes are those which are not
irreversibly inactivated or denatured when subjected to elevated
temperatures necessary for nucleic acid denaturation. Thermoactive
enzymes may or may not be thermostable. Thermoactive DNA polymerases can
be DNA or RNA dependent from thermophilic species or from mesophilic
species including, but not limited to, Escherichia coli, Moloney murine
leukemia viruses, and Avian myoblastosis virus.

[0059] As used herein, a "chimeric" protein refers to a protein whose
amino acid sequence represents a fusion product of subsequences of the
amino acid sequences from at least two distinct proteins. A chimeric
protein typically is not produced by direct manipulation of amino acid
sequences, but, rather, is expressed from a "chimeric" gene that encodes
the chimeric amino acid sequence. In certain embodiments, for example, an
unmodified form of a mutant DNA polymerase of the present invention is a
chimeric protein that consists of an amino-terminal (N-terminal) region
derived from a Thermus species DNA polymerase and a carboxy-terminal
(C-terminal) region derived from Tma DNA polymerase. The N-terminal
region refers to a region extending from the N-terminus (amino acid
position 1) to an internal amino acid. Similarly, the C-terminal region
refers to a region extending from an internal amino acid to the
C-terminus.

[0060] In the context of DNA polymerases, "correspondence" to another
sequence (e.g., regions, fragments, nucleotide or amino acid positions,
or the like) is based on the convention of numbering according to
nucleotide or amino acid position number and then aligning the sequences
in a manner that maximizes the percentage of sequence identity. Because
not all positions within a given "corresponding region" need be
identical, non-matching positions within a corresponding region may be
regarded as "corresponding positions." Accordingly, as used herein,
referral to an "amino acid position corresponding to amino acid position
[X]" of a specified DNA polymerase refers to equivalent positions, based
on alignment, in other DNA polymerases and structural homologues and
families. In some embodiments of the present invention, "correspondence"
of amino acid positions are determined with respect to a region of the
polymerase comprising one or more motifs of SEQ ID NO:1, 2, 3, 4, 5, 6,
7, 36, 37, 38, 39, 40, or 41. When a polymerase polypeptide sequence
differs from SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 36, 37, 38, 39, 40, or 41
(e.g., by changes in amino acids or addition or deletion of amino acids),
it may be that a particular mutation associated with improved activity as
discussed herein will not be in the same position number as it is in SEQ
ID NOS:1, 2, 3, 4, 5, 6, 7, 36, 37, 38, 39, 40, or 41. This is
illustrated, for example, in Table 1.

[0061] "Recombinant," as used herein, refers to an amino acid sequence or
a nucleotide sequence that has been intentionally modified by recombinant
methods. By the term "recombinant nucleic acid" herein is meant a nucleic
acid, originally formed in vitro, in general, by the manipulation of a
nucleic acid by endonucleases, in a form not normally found in nature.
Thus an isolated, mutant DNA polymerase nucleic acid, in a linear form,
or an expression vector formed in vitro by ligating DNA molecules that
are not normally joined, are both considered recombinant for the purposes
of this invention. It is understood that once a recombinant nucleic acid
is made and reintroduced into a host cell, it will replicate
non-recombinantly, i.e., using the in vivo cellular machinery of the host
cell rather than in vitro manipulations; however, such nucleic acids,
once produced recombinantly, although subsequently replicated
non-recombinantly, are still considered recombinant for the purposes of
the invention. A "recombinant protein" is a protein made using
recombinant techniques, i.e., through the expression of a recombinant
nucleic acid as depicted above.

[0062] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For example,
a promoter or enhancer is operably linked to a coding sequence if it
affects the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation.

[0063] The term "host cell" refers to both single-cellular prokaryote and
eukaryote organisms (e.g., bacteria, yeast, and actinomycetes) and single
cells from higher order plants or animals when being grown in cell
culture.

[0064] The term "vector" refers to a piece of DNA, typically
double-stranded, which may have inserted into it a piece of foreign DNA.
The vector or may be, for example, of plasmid origin. Vectors contain
"replicon" polynucleotide sequences that facilitate the autonomous
replication of the vector in a host cell. Foreign DNA is defined as
heterologous DNA, which is DNA not naturally found in the host cell,
which, for example, replicates the vector molecule, encodes a selectable
or screenable marker, or encodes a transgene. The vector is used to
transport the foreign or heterologous DNA into a suitable host cell. Once
in the host cell, the vector can replicate independently of or
coincidental with the host chromosomal DNA, and several copies of the
vector and its inserted DNA can be generated. In addition, the vector can
also contain the necessary elements that permit transcription of the
inserted DNA into an mRNA molecule or otherwise cause replication of the
inserted DNA into multiple copies of RNA. Some expression vectors
additionally contain sequence elements adjacent to the inserted DNA that
increase the half-life of the expressed mRNA and/or allow translation of
the mRNA into a protein molecule. Many molecules of mRNA and polypeptide
encoded by the inserted DNA can thus be rapidly synthesized.

[0065] The term "nucleotide," in addition to referring to the naturally
occurring ribonucleotide or deoxyribonucleotide monomers, shall herein be
understood to refer to related structural variants thereof, including
derivatives and analogs, that are functionally equivalent with respect to
the particular context in which the nucleotide is being used (e.g.,
hybridization to a complementary base), unless the context clearly
indicates otherwise.

[0066] The term "nucleic acid" or "polynucleotide" refers to a polymer
that can be corresponded to a ribose nucleic acid (RNA) or deoxyribose
nucleic acid (DNA) polymer, or an analog thereof. This includes polymers
of nucleotides such as RNA and DNA, as well as synthetic forms, modified
(e.g., chemically or biochemically modified) forms thereof, and mixed
polymers (e.g., including both RNA and DNA subunits). Exemplary
modifications include methylation, substitution of one or more of the
naturally occurring nucleotides with an analog, internucleotide
modifications such as uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, and the like), pendent
moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen,
and the like), chelators, alkylators, and modified linkages (e.g., alpha
anomeric nucleic acids and the like). Also included are synthetic
molecules that mimic polynucleotides in their ability to bind to a
designated sequence via hydrogen bonding and other chemical interactions.
Typically, the nucleotide monomers are linked via phosphodiester bonds,
although synthetic forms of nucleic acids can comprise other linkages
(e.g., peptide nucleic acids as described in Nielsen et al. (Science
254:1497-1500, 1991). A nucleic acid can be or can include, e.g., a
chromosome or chromosomal segment, a vector (e.g., an expression vector),
an expression cassette, a naked DNA or RNA polymer, the product of a
polymerase chain reaction (PCR), an oligonucleotide, a probe, and a
primer. A nucleic acid can be, e.g., single-stranded, double-stranded, or
triple-stranded and is not limited to any particular length. Unless
otherwise indicated, a particular nucleic acid sequence comprises or
encodes complementary sequences, in addition to any sequence explicitly
indicated.

[0067] The term "oligonucleotide" refers to a nucleic acid that includes
at least two nucleic acid monomer units (e.g., nucleotides). An
oligonucleotide typically includes from about six to about 175 nucleic
acid monomer units, more typically from about eight to about 100 nucleic
acid monomer units, and still more typically from about 10 to about 50
nucleic acid monomer units (e.g., about 15, about 20, about 25, about 30,
about 35, or more nucleic acid monomer units). The exact size of an
oligonucleotide will depend on many factors, including the ultimate
function or use of the oligonucleotide. Oligonucleotides are optionally
prepared by any suitable method, including, but not limited to, isolation
of an existing or natural sequence, DNA replication or amplification,
reverse transcription, cloning and restriction digestion of appropriate
sequences, or direct chemical synthesis by a method such as the
phosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99, 1979);
the phosphodiester method of Brown et al. (Meth. Enzymol. 68:109-151,
1979); the diethylphosphoramidite method of Beaucage et al. (Tetrahedron
Lett. 22:1859-1862, 1981); the triester method of Matteucci et al. (J.
Am. Chem. Soc. 103:3185-3191, 1981); automated synthesis methods; or the
solid support method of U.S. Pat. No. 4,458,066, entitled "PROCESS FOR
PREPARING POLYNUCLEOTIDES," issued Jul. 3, 1984 to Caruthers et al., or
other methods known to those skilled in the art. All of these references
are incorporated by reference.

[0068] The term "primer" as used herein refers to a polynucleotide capable
of acting as a point of initiation of template-directed nucleic acid
synthesis when placed under conditions in which polynucleotide extension
is initiated (e.g., under conditions comprising the presence of requisite
nucleoside triphosphates (as dictated by the template that is copied) and
a polymerase in an appropriate buffer and at a suitable temperature or
cycle(s) of temperatures (e.g., as in a polymerase chain reaction)). To
further illustrate, primers can also be used in a variety of other
oligonuceotide-mediated synthesis processes, including as initiators of
de novo RNA synthesis and in vitro transcription-related processes (e.g.,
nucleic acid sequence-based amplification (NASBA), transcription mediated
amplification (TMA), etc.). A primer is typically a single-stranded
oligonucleotide (e.g., oligodeoxyribonucleotide). The appropriate length
of a primer depends on the intended use of the primer but typically
ranges from 6 to 40 nucleotides, more typically from 15 to 35
nucleotides. Short primer molecules generally require cooler temperatures
to form sufficiently stable hybrid complexes with the template. A primer
need not reflect the exact sequence of the template but must be
sufficiently complementary to hybridize with a template for primer
elongation to occur. In certain embodiments, the term "primer pair" means
a set of primers including a 5' sense primer (sometimes called "forward")
that hybridizes with the complement of the 5' end of the nucleic acid
sequence to be amplified and a 3' antisense primer (sometimes called
"reverse") that hybridizes with the 3' end of the sequence to be
amplified (e.g., if the target sequence is expressed as RNA or is an
RNA). A primer can be labeled, if desired, by incorporating a label
detectable by spectroscopic, photochemical, biochemical, immunochemical,
or chemical means. For example, useful labels include 32P,
fluorescent dyes, electron-dense reagents, enzymes (as commonly used in
ELISA assays), biotin, or haptens and proteins for which antisera or
monoclonal antibodies are available.

[0069] The term "5'-nuclease probe" refers to an oligonucleotide that
comprises at least one light emitting labeling moiety and that is used in
a 5'-nuclease reaction to effect target nucleic acid detection. In some
embodiments, for example, a 5'-nuclease probe includes only a single
light emitting moiety (e.g., a fluorescent dye, etc.). In certain
embodiments, 5'-nuclease probes include regions of self-complementarity
such that the probes are capable of forming hairpin structures under
selected conditions. To further illustrate, in some embodiments a
5'-nuclease probe comprises at least two labeling moieties and emits
radiation of increased intensity after one of the two labels is cleaved
or otherwise separated from the oligonucleotide. In certain embodiments,
a 5'-nuclease probe is labeled with two different fluorescent dyes, e.g.,
a 5' terminus reporter dye and the 3' terminus quencher dye or moiety. In
some embodiments, 5'-nuclease probes are labeled at one or more positions
other than, or in addition to, terminal positions. When the probe is
intact, energy transfer typically occurs between the two fluorophores
such that fluorescent emission from the reporter dye is quenched at least
in part. During an extension step of a polymerase chain reaction, for
example, a 5'-nuclease probe bound to a template nucleic acid is cleaved
by the 5' to 3' nuclease activity of, e.g., a Taq polymerase or another
polymerase having this activity such that the fluorescent emission of the
reporter dye is no longer quenched. Exemplary 5'-nuclease probes are also
described in, e.g., U.S. Pat. No. 5,210,015, entitled "Homogeneous assay
system using the nuclease activity of a nucleic acid polymerase," issued
May 11, 1993 to Gelfand et al., U.S. Pat. No. 5,994,056, entitled
"Homogeneous methods for nucleic acid amplification and detection,"
issued Nov. 30, 1999 to Higuchi, and U.S. Pat. No. 6,171,785, entitled
"Methods and devices for homogeneous nucleic acid amplification and
detector," issued Jan. 9, 2001 to Higuchi, which are each incorporated by
reference herein. In some embodiments, a 5' nuclease probe may be labeled
with two or more different reporter dyes and a 3' terminus quencher dye
or moiety.

[0070] The term "FRET" or "fluorescent resonance energy transfer" or
"Foerster resonance energy transfer" refers to a transfer of energy
between at least two chromophores, a donor chromophore and an acceptor
chromophore (referred to as a quencher). The donor typically transfers
the energy to the acceptor when the donor is excited by light radiation
with a suitable wavelength. The acceptor typically re-emits the
transferred energy in the form of light radiation with a different
wavelength. When the acceptor is a "dark" quencher, it dissipates the
transferred energy in a form other than light. Whether a particular
fluorophore acts as a donor or an acceptor depends on the properties of
the other member of the FRET pair. Commonly used donor-acceptor pairs
include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA.
Commonly used dark quenchers include BlackHole Quenchers® (BHQ),
(Biosearch Technologies, Inc., Novato, Calif..), Iowa Black®
(Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry®
Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

[0071] The term "conventional" or "natural" when referring to nucleic acid
bases, nucleoside triphosphates, or nucleotides refers to those which
occur naturally in the polynucleotide being described (i.e., for DNA
these are dATP, dGTP, dCTP and dTTP). Additionally, dITP, and
7-deaza-dGTP are frequently utilized in place of dGTP and 7-deaza-dATP
can be utilized in place of dATP in in vitro DNA synthesis reactions,
such as sequencing. Collectively, these may be referred to as dNTPs.

[0072] The term "unconventional" or "modified" when referring to a nucleic
acid base, nucleoside, or nucleotide includes modification, derivations,
or analogues of conventional bases, nucleosides, or nucleotides that
naturally occur in a particular polynucleotide. Certain unconventional
nucleotides are modified at the 2' position of the ribose sugar in
comparison to conventional dNTPs. Thus, although for RNA the naturally
occurring nucleotides are ribonucleotides (i.e., ATP, GTP, CTP, UTP,
collectively rNTPs), because these nucleotides have a hydroxyl group at
the 2' position of the sugar, which, by comparison is absent in dNTPs, as
used herein, ribonucleotides are unconventional nucleotides as substrates
for DNA polymerases. As used herein, unconventional nucleotides include,
but are not limited to, compounds used as terminators for nucleic acid
sequencing. Exemplary terminator compounds include but are not limited to
those compounds that have a 2',3' dideoxy structure and are referred to
as dideoxynucleoside triphosphates. The dideoxynucleoside triphosphates
ddATP, ddTTP, ddCTP and ddGTP are referred to collectively as ddNTPs.
Additional examples of terminator compounds include 2'-PO4 analogs
of ribonucleotides (see, e.g., U.S. Application Publication Nos.
2005/0037991 and 2005/0037398, which are both incorporated by reference).
Other unconventional nucleotides include phosphorothioate dNTPs
([[α]-S]dNTPs), 5'-[α]-borano-dNTPs,
[α]-methyl-phosphonate dNTPs, and ribonucleoside triphosphates
(rNTPs). Unconventional bases may be labeled with radioactive isotopes
such as 32P, 33P, or 35S; fluorescent labels;
chemiluminescent labels; bioluminescent labels; hapten labels such as
biotin; or enzyme labels such as streptavidin or avidin. Fluorescent
labels may include dyes that are negatively charged, such as dyes of the
fluorescein family, or dyes that are neutral in charge, such as dyes of
the rhodamine family, or dyes that are positively charged, such as dyes
of the cyanine family. Dyes of the fluorescein family include, e.g., FAM,
HEX, TET, JOE, NAN and ZOE. Dyes of the rhodamine family include Texas
Red, ROX, R110, R6G, and TAMRA. Various dyes or nucleotides labeled with
FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA are
marketed by Perkin-Elmer (Boston, Mass.), Applied Biosystems (Foster
City, Calif.), or Invitrogen/Molecular Probes (Eugene, Oreg.). Dyes of
the cyanine family include Cy2, Cy3, Cy5, and Cy7 and are marketed by GE
Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire,
England).

[0073] As used herein, "percentage of sequence identity" is determined by
comparing two optimally aligned sequences over a comparison window,
wherein the portion of the sequence in the comparison window can comprise
additions or deletions (i.e., gaps) as compared to the reference sequence
(which does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the number
of positions at which the identical nucleic acid base or amino acid
residue occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total number
of positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity.

[0074] The terms "identical" or "identity," in the context of two or more
nucleic acids or polypeptide sequences, refer to two or more sequences or
subsequences that are the same. Sequences are "substantially identical"
to each other if they have a specified percentage of nucleotides or amino
acid residues that are the same (e.g., at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, or at least 95% identity over a
specified region), when compared and aligned for maximum correspondence
over a comparison window, or designated region as measured using one of
the following sequence comparison algorithms or by manual alignment and
visual inspection. These definitions also refer to the complement of a
test sequence. Optionally, the identity exists over a region that is at
least about 50 nucleotides in length, or more typically over a region
that is 100 to 500 or 1000 or more nucleotides in length.

[0075] The terms "similarity" or "percent similarity," in the context of
two or more polypeptide sequences, refer to two or more sequences or
subsequences that have a specified percentage of amino acid residues that
are either the same or similar as defined by a conservative amino acid
substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%,
90%, or 95% similar over a specified region), when compared and aligned
for maximum correspondence over a comparison window, or designated region
as measured using one of the following sequence comparison algorithms or
by manual alignment and visual inspection. Sequences are "substantially
similar" to each other if they are at least 20%, at least 25%, at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least
55% similar to each other. Optionally, this similarly exists over a
region that is at least about 50 amino acids in length, or more typically
over a region that is at least about 100 to 500 or 1000 or more amino
acids in length.

[0076] For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a sequence
comparison algorithm, test and reference sequences are entered into a
computer, subsequence coordinates are designated, if necessary, and
sequence algorithm program parameters are designated. Default program
parameters are commonly used, or alternative parameters can be
designated. The sequence comparison algorithm then calculates the percent
sequence identities or similarities for the test sequences relative to
the reference sequence, based on the program parameters.

[0077] A "comparison window," as used herein, includes reference to a
segment of any one of the number of contiguous positions selected from
the group consisting of from 20 to 600, usually about 50 to about 200,
more usually about 100 to about 150 in which a sequence may be compared
to a reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned. Methods of alignment of
sequences for comparison are well known in the art. Optimal alignment of
sequences for comparison can be conducted, for example, by the local
homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970),
by the homology alignment algorithm of Needleman and Wunsch (J. Mol.
Biol. 48:443, 1970), by the search for similarity method of Pearson and
Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerized
implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics Computer
Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual
inspection (see, e.g., Ausubel et al., Current Protocols in Molecular
Biology (1995 supplement)).

[0078] Algorithms suitable for determining percent sequence identity and
sequence similarity are the BLAST and BLAST 2.0 algorithms, which are
described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), and
Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software
for performing BLAST analyses is publicly available through the National
Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This
algorithm involves first identifying high scoring sequence pairs (HSPs)
by identifying short words of length W in the query sequence, which
either match or satisfy some positive-valued threshold score T when
aligned with a word of the same length in a database sequence. T is
referred to as the neighborhood word score threshold (Altschul et al.,
supra). These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are extended
in both directions along each sequence for as far as the cumulative
alignment score can be increased. Cumulative scores are calculated using,
for nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for mismatching
residues; always <0). For amino acid sequences, a scoring matrix is
used to calculate the cumulative score. Extension of the word hits in
each direction are halted when: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence is
reached. The BLAST algorithm parameters W, T, and X determine the
sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For
amino acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)
alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison
of both strands.

[0079] The BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul, Proc.
Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)),
which provides an indication of the probability by which a match between
two nucleotide or amino acid sequences would occur by chance. For
example, a nucleic acid is considered similar to a reference sequence if
the smallest sum probability in a comparison of the test nucleic acid to
the reference nucleic acid is less than about 0.2, typically less than
about 0.01, and more typically less than about 0.001.

[0080] The term "mismatch discrimination" refers to the ability of a
biocatalyst (e.g., an enzyme, such as a polymerase, ligase, or the like)
to distinguish a fully complementary sequence from a mismatch-containing
sequence when extending a nucleic acid (e.g., a primer or other
oligonucleotide) in a template-dependent manner by attaching (e.g.,
covalently) one or more nucleotides to the nucleic acid. The term
"3'-mismatch discrimination" refers to the ability of a biocatalyst to
distinguish a fully complementary sequence from a mismatch-containing
(nearly complementary) sequence where the nucleic acid to be extended
(e.g., a primer or other oligonucleotide) has a mismatch at the nucleic
acid's 3' terminus compared to the template to which the nucleic acid
hybridizes. In some embodiments, the nucleic acid to be extended
comprises a mismatch at the 3' end relative to the fully complementary
sequence. In some embodiments, the nucleic acid to be extended comprises
a mismatch at the penultimate (N-1) 3' position and/or at the N-2
position relative to the fully complementary sequence.

[0081] The term "Cp value" or "crossing point" value refers to a value
that allows quantification of input target nucleic acids. The Cp value
can be determined according to the second-derivative maximum method (Van
Luu-The, et al., "Improved real-time RT-PCR method for high-throughput
measurements using second derivative calculation and double correction,"
BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). In the second
derivative method, a Cp corresponds to the first peak of a second
derivative curve. This peak corresponds to the beginning of a log-linear
phase. The second derivative method calculates a second derivative value
of the real-time fluorescence intensity curve, and only one value is
obtained. The original Cp method is based on a locally defined,
differentiable approximation of the intensity values, e.g., by a
polynomial function. Then the third derivative is computed. The Cp value
is the smallest root of the third derivative. The Cp can also be
determined using the fit point method, in which the Cp is determined by
the intersection of a parallel to the threshold line in the log-linear
region (Van Luu-The, et al., BioTechniques, Vol. 38, No. 2, February
2005, pp. 287-293). These computations are easily carried out by any
person skilled in the art.

[0082] The term "PCR efficiency" refers to an indication of cycle to cycle
amplification efficiency for the perfectly matched primer template. PCR
efficiency is calculated for each condition using the equation: % PCR
efficiency .sub.=(10.sup.(-slope)-1)×100, where in the slope was
calculated by linear regression with the log copy number plotted on the
y-axis and Cp plotted on the x-axis.

[0083] The term "multiplex" refers to amplification with more than one set
of primers, or the amplification of more that one polymorphism site in a
single reaction.

[0086] The present invention provides improved DNA polymerases in which
one or more amino acids in the polymerase domain have been identified as
improving one or more polymerase activity or characteristics. The DNA
polymerases of the invention are active enzymes having increased
3'-mismatch discrimination activity (i.e., the inventive polymerases
described herein are less likely to extend primers that are mismatched to
template at or near the 3' end of the primer) relative to the unmodified
form of the polymerase otherwise identical except for the amino acid
difference noted herein. The DNA polymerases are useful in a variety of
applications involving polynucleotide extension or amplification of
polynucleotide templates, including, for example, applications in
recombinant DNA studies and medical diagnosis of disease.

[0094] In some embodiments, DNA polymerases of the invention derived from
a species of the Thermus genus can be characterized by having the
following motif: [0095]
Ala-Gly-His-Pro-Phe-Asn-Leu-Asn-Ser-Arg-Asp-Gln-Leu-X10-Arg-Val-Leu--
Phe-Asp-Glu-Leu (also referred to herein in the one-letter code as
A-G-H-P-F-N-L-N-S-R-D-Q-L-X10-R-V-L-F-D-E-L); [0096] wherein [0097]
X10 is any amino acid other than Glu (E) (SEQ ID NO:9).

[0098] In some embodiments, DNA polymerases of the invention derived from
a species of the Thermus genus can be characterized by having the
following motif: [0099]
Ala-Gly-His-Pro-Phe-Asn-Leu-Asn-Ser-Arg-Asp-Gln-Leu-X10-Arg-Val-Leu--
Phe-Asp-Glu-Leu (also referred to herein in the one-letter code as
A-G-H-P-F-N-L-N-S-R-D-Q-L-X10-R-V-L-F-D-E-L); [0100] wherein [0101]
X10 is Ala (A), Gly (G), Lys (K) or Arg (R) (SEQ ID NO:10).

[0102] In some embodiments, DNA polymerases of the invention derived from
a species of the Thermus genus can be characterized by having the
following motif: [0103]
Ala-Gly-His-Pro-Phe-Asn-Leu-Asn-Ser-Arg-Asp-Gln-Leu-X10-Arg-Val-Leu--
Phe-Asp-Glu-Leu (also referred to herein in the one-letter code as
A-G-H-P-F-N-L-N-S-R-D-Q-L-X10-R-V-L-F-D-E-L); [0104] wherein [0105]
X10 is Lys (K) (SEQ ID NO:11).

[0106] Further, in some embodiments, the DNA polymerases of the invention
can comprise additional amino acid substitutions, for example, at
positions X6 and X13 of the native motif (SEQ ID NO:26). The
additional substitutions at positions X6 and X13 can also
result in increased 3' mismatch discrimination. Thus, in some
embodiments, DNA polymerases of the invention can be characterized by
having the following motif: [0107]
Ala-Gly-X1-X2-Phe-X3-X4-X5-X6-X7-X.sub-
.8-Gln-X9-X10-X11-X12-Leu-X13-X14-X15-L-
eu (also referred to herein in the one-letter code as
A-G-X1-X2-F-X3-X4-X5-X6-X7-X8-Q-X-
9-X10-X11-X12-L-X13-X14-X15-L); wherein
[0108] X1 is His (H), Glu (E) or Gln (Q); [0109] X2 is Pro (P),
Thr (T) or Glu (E); [0110] X3 is Asn (N) or His (H); [0111] X4
is Leu (L) or Ile (I); [0112] X5 is Asn (N) or Arg (R); [0113]
X6 is any amino acid; [0114] X7 is Arg (R), Pro (P), or Ser
(S); [0115] X8 is Asp (D), Lys (K) or Thr (T); [0116] X9 is Leu
(L) or Val (V); [0117] X10 is any amino acid other than Glu (E), Ser
(S), Ala (A) or Gly (G); [0118] X11 is Arg (R), Asn (N), Tyr (Y),
Thr (T) or Val (V); [0119] X12 is Val (V) or Ile (I); [0120]
X13 is any amino acid; [0121] X14 is Asp (D) or Glu (E); and
[0122] X15 is Glu (E) or Lys (K) (SEQ ID NO:42)

[0123] In some embodiments, DNA polymerases of the invention can be
characterized by having the following motif: [0124]
Ala-Gly-X1-Pro-Phe-Asn-X4-Asn-X6-X7-X8-Gln-X.sub-
.9-X10-X11-X12-Leu-X13-X14-X15-Leu (also
referred to herein in the one-letter code as
A-G-X1-P-F-N-X4-N-X6-X7-X8-Q-X9-X10-X.-
sub.11-X12-L-X13-X14-X15-L); wherein [0125] X1 is
His (H) or Glu (E); [0126] X4 is Leu (L) or Ile (I); [0127] X6
is any amino acid; [0128] X7 is Arg (R) or Pro (P); [0129] X8
is Asp (D) or Lys (K); [0130] X9 is Leu (L) or Val (V); [0131]
X10 is any amino acid other than Glu (E) or Ser (S); [0132] X11
is Arg (R) or Asn (N); [0133] X12 is Val (V) or Ile (I); [0134]
X13 is any amino acid; [0135] X14 is Asp (D) or Glu (E); and
[0136] X15 is Glu (E) or Lys (K) (SEQ ID NO:43)

[0137] In some embodiments, DNA polymerases of the invention can be
characterized by having the following motif (corresponding to DNA
polymerases derived from Thermus and Thermotoga): [0138]
Ala-Gly-X1-Pro-Phe-Asn-X4-Asn-Ser-X7-X8-Gln-X9-X-
10-Arg-X12-Leu-Phe-X14-X15-Leu (also referred to
herein in the one-letter code as
A-G-X1-P-F-N-X4-N-S-X7-X8-Q-X9-X10-R-X12-L-F-X14-X15-L); wherein: [0139] X1 is His (H) or Glu
(E); [0140] X4 is Leu (L) or Ile (I); [0141] X7 is Arg (R) or
Pro (P); [0142] X8 is Asp (D) or Lys (K); [0143] X9 is Leu (L)
or Val (V); [0144] X10 is any amino acid other than Glu (E) or Ser
(S); [0145] X12 is Val (V) or Ile (I); [0146] X14 is Asp (D) or
Glu (E); and [0147] X15 is Glu (E) or Lys (K) (SEQ ID NO:44).

[0148] In some embodiments, DNA polymerases of the invention can be
characterized by having the following motif: [0149]
Ala-Gly-His-Pro-Phe-Asn-Leu-Asn-X6-Arg-Asp-Gln-Leu-X10-Arg-Val--
Leu-X13-Asp-Glu-Leu (also referred to herein in the one-letter code
as A-G-H-P-F-N-L-N-X6-R-D-Q-L-X10-R-V-L-X13-D-E-L);
wherein [0150] X6 is any amino acid; [0151] X10 is any amino
acid other than Glu (E); and [0152] X13 is any amino acid (SEQ ID
NO:45).

[0153] In some embodiments, DNA polymerases of the invention can be
characterized by having the following motif: [0154]
Ala-Gly-His-Pro-Phe-Asn-Leu-Asn-X6-Arg-Asp-Gln-Leu-X10-Arg-Val--
Leu-X13-Asp-Glu-Leu (also referred to herein in the one-letter code
as A-G-H-P-F-N-L-N-X6-R-D-Q-L-X10-R-V-L-X13-D-E-L);
wherein [0155] X6 is any amino acid other than Ser (S); [0156]
X10 is any amino acid other than Glu (E); and [0157] X13 is any
amino acid other than Phe (F) (SEQ ID NO:46).

[0171] Accordingly, in some embodiments, the invention provides for a
polymerase comprising SEQ ID NO:8, 9, 10, or 11 (e.g., where X10 is
selected, as appropriate in the consensus sequence, from G, A, V, L, I,
M, F, W, P, S, T, C, Y, N, Q, D, K, R or H) having the improved activity
and/or characteristics described herein, and wherein the DNA polymerase
is otherwise a wild-type or a naturally occurring DNA polymerase, such
as, for example, a polymerase from any of the species of thermophilic
bacteria listed above, or is substantially identical to such a wild-type
or a naturally occurring DNA polymerase. For example, in some
embodiments, the polymerase of the invention comprises SEQ ID NO:8, 9,
10, or 11 and is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:1,
2, 3, 4, 5, 6, 7, 36, 37, 38, 39, 40, or 41. In one variation, the
unmodified form of the polymerase is from a species of the genus Thermus.
In some embodiments of the invention, the unmodified polymerase is from a
thermophilic species other than Thermus, e.g., Thermotoga. The full
nucleic acid and amino acid sequence for numerous thermostable DNA
polymerases are available. The sequences each of Thermus aquaticus (Taq)
(SEQ ID NO:2), Thermus thermophilus (Tth) (SEQ ID NO:6), Thermus species
Z05 (SEQ ID NO:1), Thermus species Sps17 (SEQ ID NO:5), Thermotoga
maritima (Tma) (SEQ ID NO:38), and Thermosipho africanus (Taf) (SEQ ID
NO:37) polymerase have been published in PCT International Patent
Publication No. WO 92/06200, which is incorporated herein by reference.
The sequence for the DNA polymerase from Thermus flavus (SEQ ID NO:4) has
been published in Akhmetzjanov and Vakhitov (Nucleic Acids Research
20:5839, 1992), which is incorporated herein by reference. The sequence
of the thermostable DNA polymerase from Thermus caldophilus (SEQ ID NO:7)
is found in EMBL/GenBank Accession No. U62584. The sequence of the
thermostable DNA polymerase from Thermus filiformis can be recovered from
ATCC Deposit No. 42380 using, e.g., the methods provided in U.S. Pat. No.
4,889,818, as well as the sequence information provided in Table 1. The
sequence of the Thermotoga neapolitana DNA polymerase (SEQ ID NO:39) is
from GeneSeq Patent Data Base Accession No. R98144 and PCT WO 97/09451,
each incorporated herein by reference. The sequence of the thermostable
DNA polymerase from Bacillus caldotenax (SEQ ID NO:41) is described in,
e.g., Uemori et al. (J Biochem (Tokyo) 113(3):401-410, 1993; see also,
Swiss-Prot database Accession No. Q04957 and GenBank Accession Nos.
D12982 and BAA02361), which are each incorporated by reference. Examples
of unmodified forms of DNA polymerases that can be modified as described
herein are also described in, e.g., U.S. Pat. No. 6,228,628, entitled
"Mutant chimeric DNA polymerase" issued May 8, 2001 to Gelfand et al.;
U.S. Pat. No. 6,346,379, entitled "Thermostable DNA polymerases
incorporating nucleoside triphosphates labeled with fluorescein family
dyes" issued Feb. 12, 2002 to Gelfand et al.; U.S. Pat. No. 7,030,220,
entitled "Thermostable enzyme promoting the fidelity of thermostable DNA
polymerases-for improvement of nucleic acid synthesis and amplification
in vitro" issued Apr. 18, 2006 to Ankenbauer et al.; U.S. Pat. No.
6,881,559, entitled "Mutant B-type DNA polymerases exhibiting improved
performance in PCR" issued Apr. 19, 2005 to Sobek et al.; U.S. Pat. No.
6,794,177, entitled "Modified DNA-polymerase from carboxydothermus
hydrogenoformans and its use for coupled reverse transcription and
polymerase chain reaction" issued Sep. 21, 2004 to Markau et al.; U.S.
Pat. No. 6,468,775, entitled "Thermostable DNA polymerase from
carboxydothermus hydrogenoformans" issued Oct. 22, 2002 to Ankenbauer et
al.; and U.S. Pat. Appl. Nos. 20040005599, entitled "Thermostable or
thermoactive DNA polymerase molecules with attenuated 3'-5' exonuclease
activity" filed Mar. 26, 2003 by Schoenbrunner et al.; 20020012970,
entitled "High temperature reverse transcription using mutant DNA
polymerases" filed Mar. 30, 2001 by Smith et al.; 20060078928, entitled
"Thermostable enzyme promoting the fidelity of thermostable DNA
polymerases-for improvement of nucleic acid synthesis and amplification
in vitro" filed Sep. 29, 2005 by Ankenbauer et al.; 20040115639, entitled
"Reversibly modified thermostable enzymes for DNA synthesis and
amplification in vitro" filed Dec. 11, 2002 by Sobek et al., which are
each incorporated by reference. Representative full length polymerase
sequences are also provided in the sequence listing.

[0172] In some embodiments, the polymerase of the invention, as well as
having a polymerase domain comprising SEQ ID NOS:8, 9, 10, or 11, also
comprises a nuclease domain (e.g., corresponding to positions 1 to 291 of
Z05).

[0173] In some embodiments, a polymerase of the invention is a chimeric
polymerase, i.e., comprising polypeptide regions from two or more
enzymes. Examples of such chimeric DNA polymerases are described in,
e.g., U.S. Pat. No. 6,228,628, which is incorporated by reference herein
in its entirety. Particularly suitable are chimeric CS-family DNA
polymerases, which include the CS5 (SEQ ID NO:29) and CS6 (SEQ ID NO:30)
polymerases and variants thereof having substantial sequence identity or
similarity to SEQ ID NO:29 or SEQ ID NO:30 (typically at least 80%
sequence identity and more typically at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% sequence identity) and can thus be modified to
contain SEQ ID NO:8. The CS5 and CS6 DNA polymerases are chimeric enzymes
derived from Thermus sp. Z05 and Thermotoga maritima (Tma) DNA
polymerases. They comprise the N-terminal 5'-nuclease domain of the
Thermus enzyme and the C-terminal 3'-5' exonuclease and the polymerase
domains of the Tma enzyme. These enzymes have efficient reverse
transcriptase activity, can extend nucleotide analog-containing primers,
and can utilize alpha-phosphorothioate dNTPs, dUTP, dITP, and also
fluorescein- and cyanine-dye family labeled dNTPs. The CS5 and CS6
polymerases are also efficient Mg2+-activated PCR enzymes. The CS5
and CS6 chimeric polymerases are further described in, e.g., U.S. Pat.
Application Publication No. 2004/0005599, which is incorporated by
reference herein in its entirety.

[0174] In some embodiments, the polymerase of the invention comprises SEQ
ID NO:8, 9, 10, or 11 and further comprises one or more additional amino
acid changes (e.g., by amino acid substitution, addition, or deletion)
compared to a native polymerase. In some embodiments, such polymerases
retain the amino acid motif of SEQ ID NO:8 (or a motif of SEQ ID NO:9, 10
or 11), and further comprise the amino acid motif of SEQ ID NO:27
(corresponding to the D580X mutation of Z05 (SEQ ID NO:1)) as follows:
[0175] T-G-R-L-S-S-X7-X8-P-N-L-Q-N; wherein [0176] X7 is
Ser (S) or Thr (T); and [0177] X8 is any amino acid other than D or
E (SEQ ID NO:27) The mutation characterized by SEQ ID NO:27 is discussed
in more detail in, e.g., U.S. Patent Publication No. 2009/0148891. In
some embodiments, such functional variant polymerases typically will have
substantial sequence identity or similarity to the wild-type or naturally
occurring polymerase (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 39, 40, 41,
42, 43, or 44), typically at least 80% sequence identity and more
typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
sequence identity.

[0178] In some embodiments, the amino acid at position X10 is
substituted with an amino acid as set forth in SEQ ID NO:8, 9, 10 or 11,
and the amino acid at position X8 is substituted with an amino acid
as set forth in SEQ ID NO:27. Thus, in some embodiments, the amino acid
at position X10 is any amino acid other than Glu (E), Ser (5), Ala
(A),or Gly (G), and the amino acid at position X8 is any amino acid
other than Asp (D) or Glu (E). In some embodiments, amino acid
substitutions include Leucine (L), Glycine (G), Threonine (T), Glutamine
(Q), Alanine (A), Serine (S), Asparagine (N), Arginine (R), and Lysine
(K) at position X8 of SEQ ID NO:27. In certain embodiments, where
the DNA polymerase of the invention is derived from Thermus sp., amino
acid substitutions independently include Alanine (A), Glycine (G), Lysine
(K), or Arginine (R)at position X10 and Glycine (G) at position
X8. In certain embodiments, amino acid substitutions independently
include Lysine (K) at position X10, and Glycine (G) at position
X8. Other suitable amino acid substitution(s) at one or more of the
identified sites can be determined using, e.g., known methods of
site-directed mutagenesis and determination of polynucleotide extension
performance in assays described further herein or otherwise known to
persons of skill in the art.

[0179] Because the precise length of DNA polymerases vary, the precise
amino acid positions corresponding to each of X10 and X8 can
vary depending on the particular polymerase used. Amino acid and nucleic
acid sequence alignment programs are readily available (see, e.g., those
referred to supra) and, given the particular motifs identified herein,
serve to assist in the identification of the exact amino acids (and
corresponding codons) for modification in accordance with the present
invention. The positions corresponding to each of X6, X10,
X13 and X8 are shown in Table 1 for representative chimeric
thermostable DNA polymerases and thermostable DNA polymerases from
exemplary thermophilic species.

[0180] In some embodiments, the mutant DNA polymerase of the present
invention is derived from Thermus sp. Z05 DNA polymerase (SEQ ID NO:1) or
a variant thereof (e.g., D580G or the like). As referred to above, in
native Thermus sp. Z05 DNA polymerase, position X10 corresponds to
Glutamic acid (E) at position 493; position X6 corresponds to Serine
(S) at position 488; position X13 corresponds to Phenylalanine (F)
at position 497 of SEQ ID NO:1; and position X8 of SEQ ID NO:27
corresponds to Aspartate (D) at position 580 of SEQ ID NO:1. Thus, in
certain variations of the invention, the mutant polymerase comprises at
least one amino acid substitution, relative to a Thermus sp. Z05 DNA
polymerase, at E493, 5488, F497, and D580. Thus, in some embodiments, the
amino acid at position E493, is not E. In some embodiments, the amino
acid at position 493 is selected from G, A, V, L, I, M, F, W, P, S, T, C,
Y, N, Q, D, K, R or H. In certain embodiments, amino acid residue at
position E493 is S, A, Q, G, K or R. Typically, the amino acid at
position 5488, if substituted, is not S. In some embodiments, the amino
acid at position 488 of SEQ ID NO:1 is selected from G, A, V, L, I, M, F,
W, P, E, T, C, N, Q, D, Y, K, R or H. In certain embodiments, the amino
acid residue at position S488 can be substituted or not substituted, and
is S, G, A, D, F, K, C, T, or Y. Typically, the amino acid at position
F497, if substituted, is not F. In some embodiments, the amino acid at
position 497 is selected from G, A, V, L, I, M, E, W, P, S, T, C, Y, N,
Q, D, K, R or H. In certain embodiments, the amino acid residue at
position F497 can be substituted or not substituted, and is F, A, G, S,
T, Y, D, or K. In certain embodiments, amino acid residues at position
D580 can be selected from Leucine (L), Glycine (G), Threonine (T),
Glutamine (Q), Alanine (A), Serine (S), Asparagine (N), Arginine (R), and
Lysine (K). Exemplary Thermus sp. Z05 DNA polymerase mutants include
those comprising the amino acid substitution(s) E493K and D580G.

[0182] In some embodiments, the DNA polymerase of the invention is derived
from a Thermus species, and the amino acid corresponding to position 493
of SEQ ID NO:1 is an amino acid that does not have a polar,
negatively-charged side-chain at neutral pH (e.g., E). Thus, in some
embodiments, the DNA polymerase of the invention is derived from a
Thermus species, and the amino acid corresponding to position 493 of SEQ
ID NO:1 has a polar, uncharged side-chain (e.g., S, Q), a nonpolar,
uncharged side-chain (e.g., A, G), or a polar, positively charged
side-chain (e.g., R or K) at neutral pH (e.g., about pH 7.4). In some
embodiments, the amino acid corresponding to position 493 of SEQ ID NO:1
has a nonpolar, uncharged side-chain (e.g., A, G), or a polar, positively
charged side-chain (e.g., R or K). In some embodiments, the amino acid
corresponding to position 493 of SEQ ID NO:1 having a polar, positively
charged side-chain is R or K. In some embodiments, the amino acid
corresponding to position 493 of SEQ ID NO:1 having a polar, positively
charged side-chain is K.

[0183] In some embodiments, the DNA polymerases of the present invention
can also include other, non-substitutional modification(s). Such
modifications can include, for example, covalent modifications known in
the art to confer an additional advantage in applications comprising
polynucleotide extension. For example, in certain embodiments, the mutant
DNA polymerase further includes a thermally reversible covalent
modification. DNA polymerases comprising such thermally reversible
modifications are particularly suitable for hot-start applications, such
as, e.g., various hot-start PCR techniques. Thermally reversible modifier
reagents amenable to use in accordance with the mutant DNA polymerases of
the present invention are described in, for example, U.S. Pat. No.
5,773,258 to Birch et al., which is incorporated by reference herein.

[0184] For example, particularly suitable polymerases comprising a
thermally reversible covalent modification are produced by a reaction,
carried out at alkaline pH at a temperature which is less than about
25° C., of a mixture of a thermostable enzyme and a dicarboxylic
acid anhydride having a general foimula as set forth in the following
formula I:

##STR00001##

where R1 and R2 are hydrogen or organic radicals, which may be
linked; or having the following formula II:

##STR00002##

where R1 and R2 are organic radicals, which may linked, and the
hydrogens are cis, essentially as described in Birch et al, supra.

[0185] The DNA polymerases of the present invention can be constructed by
mutating the DNA sequences that encode the corresponding unmodified
polymerase (e.g., a wild-type polymerase or a corresponding variant from
which the polymerase of the invention is derived), such as by using
techniques commonly referred to as site-directed mutagenesis. Nucleic
acid molecules encoding the unmodified foam of the polymerase can be
mutated by a variety of polymerase chain reaction (PCR) techniques
well-known to one of ordinary skill in the art. (See, e.g., PCR
Strategies (M. A. Innis, D. H. Gelfand, and J. J. Sninsky eds., 1995,
Academic Press, San Diego, Calif.) at Chapter 14; PCR Protocols: A Guide
to Methods and Applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky,
and T. J. White eds., Academic Press, NY, 1990).

[0186] By way of non-limiting example, the two primer system, utilized in
the Transformer Site-Directed Mutagenesis kit from Clontech, may be
employed for introducing site-directed mutants into a polynucleotide
encoding an unmodified form of the polymerase. Following denaturation of
the target plasmid in this system, two primers are simultaneously
annealed to the plasmid; one of these primers contains the desired
site-directed mutation, the other contains a mutation at another point in
the plasmid resulting in elimination of a restriction site. Second strand
synthesis is then carried out, tightly linking these two mutations, and
the resulting plasmids are transformed into a mutS strain of E. coli.
Plasmid DNA is isolated from the transformed bacteria, restricted with
the relevant restriction enzyme (thereby linearizing the unmutated
plasmids), and then retransformed into E. coli. This system allows for
generation of mutations directly in an expression plasmid, without the
necessity of subcloning or generation of single-stranded phagemids. The
tight linkage of the two mutations and the subsequent linearization of
unmutated plasmids result in high mutation efficiency and allow minimal
screening Following synthesis of the initial restriction site primer,
this method requires the use of only one new primer type per mutation
site. Rather than prepare each positional mutant separately, a set of
"designed degenerate" oligonucleotide primers can be synthesized in order
to introduce all of the desired mutations at a given site simultaneously.
Transformants can be screened by sequencing the plasmid DNA through the
mutagenized region to identify and sort mutant clones. Each mutant DNA
can then be restricted and analyzed by electrophoresis, such as for
example, on a Mutation Detection Enhancement gel (Mallinckrodt Baker,
Inc., Phillipsburg, N.J.) to confirm that no other alterations in the
sequence have occurred (by band shift comparison to the unmutagenized
control). Alternatively, the entire DNA region can be sequenced to
confirm that no additional mutational events have occurred outside of the
targeted region.

[0187] Verified mutant duplexes in pET (or other) overexpression vectors
can be employed to transform E. coli such as, e.g., strain E. coli BL21
(DE3) pLysS, for high level production of the mutant protein, and
purification by standard protocols. The method of FAB-MS mapping, for
example, can be employed to rapidly check the fidelity of mutant
expression. This technique provides for sequencing segments throughout
the whole protein and provides the necessary confidence in the sequence
assignment. In a mapping experiment of this type, protein is digested
with a protease (the choice will depend on the specific region to be
modified since this segment is of prime interest and the remaining map
should be identical to the map of unmutagenized protein). The set of
cleavage fragments is fractionated by, for example, microbore HPLC
(reversed phase or ion exchange, again depending on the specific region
to be modified) to provide several peptides in each fraction, and the
molecular weights of the peptides are determined by standard methods,
such as FAB-MS. The determined mass of each fragment are then compared to
the molecular weights of peptides expected from the digestion of the
predicted sequence, and the correctness of the sequence quickly
ascertained. Since this mutagenesis approach to protein modification is
directed, sequencing of the altered peptide should not be necessary if
the MS data agrees with prediction. If necessary to verify a changed
residue, CAD-tandem MS/MS can be employed to sequence the peptides of the
mixture in question, or the target peptide can be purified for
subtractive Edman degradation or carboxypeptidase Y digestion depending
on the location of the modification.

[0188] Mutant DNA polymerases with more than one amino acid substituted
can be generated in various ways. In the case of amino acids located
close together in the polypeptide chain, they may be mutated
simultaneously using one oligonucleotide that codes for all of the
desired amino acid substitutions. If however, the amino acids are located
some distance from each other (separated by more than ten amino acids,
for example) it is more difficult to generate a single oligonucleotide
that encodes all of the desired changes. Instead, one of two alternative
methods may be employed. In the first method, a separate oligonucleotide
is generated for each amino acid to be substituted. The oligonucleotides
are then annealed to the single-stranded template DNA simultaneously, and
the second strand of DNA that is synthesized from the template will
encode all of the desired amino acid substitutions. An alternative method
involves two or more rounds of mutagenesis to produce the desired mutant.
The first round is as described for the single mutants: DNA encoding the
unmodified polymerase is used for the template, an oligonucleotide
encoding the first desired amino acid substitution(s) is annealed to this
template, and the heteroduplex DNA molecule is then generated. The second
round of mutagenesis utilizes the mutated DNA produced in the first round
of mutagenesis as the template. Thus, this template already contains one
or more mutations. The oligonucleotide encoding the additional desired
amino acid substitution(s) is then annealed to this template, and the
resulting strand of DNA now encodes mutations from both the first and
second rounds of mutagenesis. This resultant DNA can be used as a
template in a third round of mutagenesis, and so on. Alternatively, the
multi-site mutagenesis method of Seyfang & Jin (Anal. Biochem.
324:285-291. 2004) may be utilized.

[0189] Accordingly, also provided are recombinant nucleic acids encoding
any of the DNA polymerases of the present invention (e.g., polymerases
comprising any of SEQ ID NOS:8, 9, 10, or 11). Using a nucleic acid of
the present invention, encoding a DNA polymerase of the invention, a
variety of vectors can be made. Any vector containing replicon and
control sequences that are derived from a species compatible with the
host cell can be used in the practice of the invention. Generally,
expression vectors include transcriptional and translational regulatory
nucleic acid regions operably linked to the nucleic acid encoding the
mutant DNA polymerase. The term "control sequences" refers to DNA
sequences necessary for the expression of an operably linked coding
sequence in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter, optionally an
operator sequence, and a ribosome binding site. In addition, the vector
may contain a Positive Retroregulatory Element (PRE) to enhance the
half-life of the transcribed mRNA (see Gelfand et al. U.S. Pat. No.
4,666,848). The transcriptional and translational regulatory nucleic acid
regions will generally be appropriate to the host cell used to express
the polymerase. Numerous types of appropriate expression vectors, and
suitable regulatory sequences are known in the art for a variety of host
cells. In general, the transcriptional and translational regulatory
sequences may include, e.g., promoter sequences, ribosomal binding sites,
transcriptional start and stop sequences, translational start and stop
sequences, and enhancer or activator sequences. In typical embodiments,
the regulatory sequences include a promoter and transcriptional start and
stop sequences. Vectors also typically include a polylinker region
containing several restriction sites for insertion of foreign DNA. In
certain embodiments, "fusion flags" are used to facilitate purification
and, if desired, subsequent removal of tag/flag sequence, e.g.,
"His-Tag". However, these are generally unnecessary when purifying an
thermoactive and/or thermostable protein from a mesophilic host (e.g., E.
coli) where a "heat-step" may be employed. The construction of suitable
vectors containing DNA encoding replication sequences, regulatory
sequences, phenotypic selection genes, and the mutant polymerase of
interest are prepared using standard recombinant DNA procedures. Isolated
plasmids, viral vectors, and DNA fragments are cleaved, tailored, and
ligated together in a specific order to generate the desired vectors, as
is well-known in the art (see, e.g., Sambrook et al., Molecular Cloning:
A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, N.Y.,
2nd ed. 1989)).

[0190] In certain embodiments, the expression vector contains a selectable
marker gene to allow the selection of transformed host cells. Selection
genes are well known in the art and will vary with the host cell used.
Suitable selection genes can include, for example, genes coding for
ampicillin and/or tetracycline resistance, which enables cells
transformed with these vectors to grow in the presence of these
antibiotics.

[0191] In one aspect of the present invention, a nucleic acid encoding a
DNA polymerase of the invention is introduced into a cell, either alone
or in combination with a vector. By "introduced into" or grammatical
equivalents herein is meant that the nucleic acids enter the cells in a
manner suitable for subsequent integration, amplification, and/or
expression of the nucleic acid. The method of introduction is largely
dictated by the targeted cell type. Exemplary methods include CaPO4
precipitation, liposome fusion, LIPOFECTIN®, electroporation, viral
infection, and the like.

[0192] In some embodiments, prokaryotes are used as host cells for the
initial cloning steps of the present invention. They are particularly
useful for rapid production of large amounts of DNA, for production of
single-stranded DNA templates used for site-directed mutagenesis, for
screening many mutants simultaneously, and for DNA sequencing of the
mutants generated. Suitable prokaryotic host cells include E. coli K12
strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325), E.
coli K12 strain DG116 (ATCC No. 53,606), E. coli X1776 (ATCC No. 31,537),
and E. coli B; however many other strains of E. coli, such as HB101,
JM101, NM522, NM538, NM539, and many other species and genera of
prokaryotes including bacilli such as Bacillus subtilis, other
enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans,
and various Pseudomonas species can all be used as hosts. Prokaryotic
host cells or other host cells with rigid cell walls are typically
transformed using the calcium chloride method as described in section
1.82 of Sambrook et al., supra. Alternatively, electroporation can be
used for transformation of these cells. Prokaryote transformation
techniques are set forth in, for example Dower, in Genetic Engineering,
Principles and Methods 12:275-296 (Plenum Publishing Corp., 1990);
Hanahan et al., Meth. Enzymol., 204:63, 1991. Plasmids typically used for
transformation of E. coli include pBR322, pUCI8, pUCI9, pUCI18, pUC119,
and Bluescript M13, all of which are described in sections 1.12-1.20 of
Sambrook et al., supra. However, many other suitable vectors are
available as well.

[0193] In some embodiments, the DNA polymerases of the present invention
are produced by culturing a host cell transformed with an expression
vector containing a nucleic acid encoding the DNA polymerase, under the
appropriate conditions to induce or cause expression of the DNA
polymerase. Methods of culturing transformed host cells under conditions
suitable for protein expression are well-known in the art (see, e.g.,
Sambrook et al., supra). Suitable host cells for production of the
polymerases from lambda pL promotor-containing plasmid vectors include E.
coli strain DG116 (ATCC No. 53606) (see U.S. Pat. No. 5,079,352 and
Lawyer, F. C. et al., PCR Methods and Applications 2:275-87, 1993, which
are both incorporated herein, by reference). Following expression, the
polymerase can be harvested and isolated. Methods for purifying the
thermostable DNA polymerase are described in, for example, Lawyer et al.,
supra.

[0194] Once purified, a DNA polymerase's 3' mismatch discrimination can be
assayed. For example, in some embodiments, 3' mismatch discrimination
activity is determined by comparing the amplification of a target
sequence perfectly matched to the primer to amplification of a target
that has a single base mismatch at the 3' end of the primer.
Amplification can be detected, for example, in real time by use of
TaqMan® probes. Ability of a polymerase to distinguish between the two
target sequences can be estimated by comparing the Cps of the two
reactions. Optionally, simultaneous amplification of a second target gene
in each well can be performed and detected in a second optical channel as
a control. "Delta Cp values" refer to the difference in value between the
Cp associated with the mismatched template minus the Cp of the matched
target (see, e.g., the Examples). In some embodiments, the improved
polymerases of the invention have a delta Cp value of at least 1, 2, 3,
4, 5, or more compared to an otherwise identical control polymerase
having a native amino acid (e.g., E) at position X10 of SEQ ID NO:8.
In some embodiments, this determination is made with the precise
materials and conditions set forth in the Examples.

Methods of the Invention

[0195] The improved DNA polymerases of the present invention may be used
for any purpose in which such enzyme activity is necessary or desired.
The improved DNA polymerase can be a thermoactive or thermostable DNA
polymerase, as described herein. Accordingly, in one aspect of the
invention, methods of polynucleotide extension, including PCR, using the
polymerases of the invention are provided. In some embodiments, the
invention provides a thermoactive DNA polymerase that is useful to extend
an RNA or DNA template when amplification of the template nucleic acid is
not required, for example, when it is desired to immediately detect the
presence of a target nucleic acid. In some embodiments, the invention
provides a thermostable DNA polymerase that is useful when it is desired
to extend and/or amplify a target nucleic acid. Conditions suitable for
polynucleotide extension are known in the art. (See, e.g., Sambrook et
al., supra. See also Ausubel et al., Short Protocols in Molecular Biology
(4th ed., John Wiley & Sons 1999). Generally, a primer is annealed, i.e.,
hybridized, to a target nucleic acid to form a primer-template complex.
The primer-template complex is contacted with the mutant DNA polymerase
and nucleoside triphosphates in a suitable environment to permit the
addition of one or more nucleotides to the 3' end of the primer, thereby
producing an extended primer complementary to the target nucleic acid.
The primer can include, e.g., one or more nucleotide analog(s). In
addition, the nucleoside triphosphates can be conventional nucleotides,
unconventional nucleotides (e.g., ribonucleotides or labeled
nucleotides), or a mixture thereof In some variations, the polynucleotide
extension reaction comprises amplification of a target nucleic acid.
Conditions suitable for nucleic acid amplification using a DNA polymerase
and a primer pair are also known in the art (e.g., PCR amplification
methods). (See, e.g., Sambrook et al., supra; Ausubel et al., supra; PCR
Applications: Protocols for Functional Genomics (Innis et al. eds.,
Academic Press 1999).

[0196] In some embodiments, use of the present polymerases, which provide
increased 3' mismatch discrimination, allow for, e.g., rare allele
detection. For example, the fidelity of 3' mismatch discrimination of a
particular polymerase sets its sensitivity (ability to accurately detect
small quantities of a target sequence in the presence of larger
quantities of a different but related non-target sequence). Thus,
increased 3'-mismatch discrimination results in greater sensitivity for
detection of rare alleles. Rare allele detection is useful, for example,
when screening biopsies or other samples for rare genetic changes, e.g.,
a cell carrying a cancer allele in a mass of normal cells.

[0198] Detection of multiple different alleles can also be accomplished
using multiplex reactions, which allow the detection of multiple
different alleles in a single reaction. In multiplex reactions, two or
more allele-specific primers are used to extend and amplify SNPs or
multiple nucleotide polymorphisms or alleles. Exemplary methods for
multiplex detection of single and multiple nucleotide polymorphisms are
described in U.S. Patent Publication No. 2006/0172324, the contents of
which are expressly incorporated by reference herein in its entirety.

[0199] Other methods for detecting extension products or amplification
products using the improved polymerases described herein include the use
of fluorescent double-stranded nucleotide binding dyes or fluorescent
double-stranded nucleotide intercalating dyes. Examples of fluorescent
double-stranded DNA binding dyes include SYBR-green (Molecular Probes).
Examples of fluorescent double-stranded intercalating dyes include
ethidium bromide. The double stranded DNA binding dyes can be used in
conjunction with melting curve analysis to measure primer extension
products and/or amplification products. The melting curve analysis can be
performed on a real-time PCR instrument, such as the ABI 5700/7000 (96
well format) or ABI 7900 (384 well format) instrument with onboard
software (SDS 2.1). Alternatively, the melting curve analysis can be
performed as an end point analysis. Exemplary methods of melting point
analysis are described in U.S. Patent Publication No. 2006/0172324, the
contents of which are expressly incorporated by reference herein in its
entirety.

[0200] In yet other embodiments, the polymerases of the invention are used
for primer extension in the context of DNA sequencing, DNA labeling, or
labeling of primer extension products. For example, DNA sequencing by the
Sanger dideoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci.
USA 74: 5463, 1977) is improved by the present invention for polymerases
capable of incorporating unconventional, chain-terminating nucleotides.
Advances in the basic Sanger et al. method have provided novel vectors
(Yanisch-Perron et al., Gene 33:103-119, 1985) and base analogues (Mills
et al., Proc. Natl. Acad. Sci. USA 76:2232-2235, 1979; and Barr et al.,
Biotechniques 4:428-432, 1986). In general, DNA sequencing requires
template-dependent primer extension in the presence of chain-terminating
base analogs, resulting in a distribution of partial fragments that are
subsequently separated by size. The basic dideoxy sequencing procedure
involves (i) annealing an oligonucleotide primer, optionally labeled, to
a template; (ii) extending the primer with DNA polymerase in four
separate reactions, each containing a mixture of unlabeled dNTPs and a
limiting amount of one chain terminating agent such as a ddNTP,
optionally labeled; and (iii) resolving the four sets of reaction
products on a high-resolution denaturing polyacrylamide/urea gel. The
reaction products can be detected in the gel by autoradiography or by
fluorescence detection, depending on the label used, and the image can be
examined to infer the nucleotide sequence. These methods utilize DNA
polymerase such as the Klenow fragment of E. coli Pol I or a modified T7
DNA polymerase.

[0201] The availability of thermostable polymerases, such as Taq DNA
polymerase, resulted in improved methods for sequencing with thermostable
DNA polymerase (see Innis et al., Proc. Natl. Acad. Sci. USA 85:9436,
1988) and modifications thereof referred to as "cycle sequencing"
(Murray, Nuc Acids Res. 17:8889, 1989). Accordingly, thermostable
polymerases of the present invention can be used in conjunction with such
methods. As an alternative to basic dideoxy sequencing, cycle sequencing
is a linear, asymmetric amplification of target sequences complementary
to the template sequence in the presence of chain terminators. A single
cycle produces a family of extension products of all possible lengths.
Following denaturation of the extension reaction product from the DNA
template, multiple cycles of primer annealing and primer extension occur
in the presence of terminators such as ddNTPs. Cycle sequencing requires
less template DNA than conventional chain-termination sequencing.
Thermostable DNA polymerases have several advantages in cycle sequencing;
they tolerate the stringent annealing temperatures which are required for
specific hybridization of primer to nucleic acid targets as well as
tolerating the multiple cycles of high temperature denaturation which
occur in each cycle, e.g., 90-95° C. For this reason,
AMPLITAQ® DNA Polymerase and its derivatives and descendants, e.g.,
AmpliTaq CS DNA Polymerase and AmpliTaq FS DNA Polymerase have been
included in Taq cycle sequencing kits commercialized by companies such as
Perkin-Elmer (Norwalk, Conn.) and Applied Biosystems (Foster City,
Calif.).

[0202] The improved polymerases find use in 454 sequencing (Roche)
(Margulies, M et al. 2005, Nature, 437, 376-380). 454 sequencing involves
two steps. In the first step, DNA is sheared into fragments of
approximately 300-800 base pairs, and the fragments are blunt ended.
Oligonucleotide adaptors are then ligated to the ends of the fragments.
The adaptors serve as primers for amplification and sequencing of the
fragments. The fragments can be attached to DNA capture beads, e.g.,
streptavidin-coated beads using, e.g., Adaptor B, which contains
5'-biotin tag. The fragments attached to the beads are PCR amplified
within droplets of an oil-water emulsion. The result is multiple copies
of clonally amplified DNA fragments on each bead. In the second step, the
beads are captured in wells (pico-liter sized). Pyrosequencing is
performed on each DNA fragment in parallel. Addition of one or more
nucleotides generates a light signal that is recorded by a CCD camera in
a sequencing instrument. The signal strength is proportional to the
number of nucleotides incorporated.

[0203] Pyrosequencing makes use of pyrophosphate (PPi) which is released
upon nucleotide addition. PPi is converted to ATP by ATP sulfurylase in
the presence of adenosine 5' phosphosulfate. Luciferase uses ATP to
convert luciferin to oxyluciferin, and this reaction generates light that
is detected and analyzed.

[0204] Variations of chain termination sequencing methods include
dye-primer sequencing and dye-terminator sequencing. In dye-primer
sequencing, the ddNTP terminators are unlabeled, and a labeled primer is
utilized to detect extension products (Smith et al., Nature 32:674-679,
1986). In dye-terminator DNA sequencing, a DNA polymerase is used to
incorporate dNTPs and fluorescently labeled ddNTPs onto the end of a DNA
primer (Lee et al., Nuc. Acids. Res. 20:2471, 1992). This process offers
the advantage of not having to synthesize dye labeled primers.
Furthermore, dye-terminator reactions are more convenient in that all
four reactions can be performed in the same tube.

[0205] Both dye-primer and dye-terminator methods may be automated using
an automated sequencing instrument produced by Applied Biosystems (Foster
City, Calif.) (U.S. Pat. No. 5,171,534, which is herein incorporated by
reference). When using the instrument, the completed sequencing reaction
mixture is fractionated on a denaturing polyacrylamide gel or capillaries
mounted in the instrument. A laser at the bottom of the instrument
detects the fluorescent products as they are electrophoretically
separated according to size through the gel.

[0206] Two types of fluorescent dyes are commonly used to label the
terminators used for dye-terminator sequencing-negatively charged and
zwitterionic fluorescent dyes. Negatively charged fluorescent dyes
include those of the fluorescein and BODIPY families. BODIPY dyes
(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) are described in
International Patent Publication WO 97/00967, which is incorporated
herein by reference. Zwitterionic fluorescent dyes include those of the
rhodamine family. Commercially available cycle sequencing kits use
terminators labeled with rhodamine derivatives. However, the
rhodamine-labeled terminators are rather costly and the product must be
separated from unincorporated dye-ddNTPs before loading on the gel since
they co-migrate with the sequencing products. Rhodamine dye family
terminators seem to stabilize hairpin structures in GC-rich regions,
which causes the products to migrate anomalously. This can involve the
use of dITP, which relaxes the secondary structure but also affects the
efficiency of incorporation of terminator.

[0207] In contrast, fluorescein-labeled terminators eliminate the
separation step prior to gel loading since they have a greater net
negative charge and migrate faster than the sequencing products. In
addition, fluorescein-labeled sequencing products have better
electrophoretic migration than sequencing products labeled with
rhodamine. Although wild-type Taq DNA polymerase does not efficiently
incorporate terminators labeled with fluorescein family dyes, this can
now be accomplished efficiently by use of the modified enzymes as
described in U.S. Patent Application Publication No. 2002/0142333, which
is incorporated by reference herein in its entirety. Accordingly,
modifications as described in U.S. 2002/0142333 can be used in the
context of the present invention to produce
fluorescein-family-dye-incorporating thermostable polymerases having
improved primer extension rates. For example, in certain embodiments, the
unmodified DNA polymerase in accordance with the present invention is a
modified thermostable polymerase as described in U.S. 2002/0142333 and
having the motif set forth in SEQ ID NO:8 (or a motif of SEQ ID NO:9, 10
or 11), and optionally the motif of SEQ ID NO:27.

[0208] Other exemplary nucleic acid sequencing formats in which the mutant
DNA polymerases of the invention can be used include those involving
terminator compounds that include 2'-PO4 analogs of ribonucleotides
(see, e.g., U.S. Application Publication Nos. 2005/0037991 and
2005/0037398, and U.S. patent application Ser. No. 12/174,488, which are
each incorporated by reference).

Kits

[0209] In another aspect of the present invention, kits are provided for
use in primer extension methods described herein. In some embodiments,
the kit is compartmentalized for ease of use and contains at least one
container providing a DNA polymerase of the invention having increased 3'
mismatch discrimination in accordance with the present invention. One or
more additional containers providing additional reagent(s) can also be
included. Such additional containers can include any reagents or other
elements recognized by the skilled artisan for use in primer extension
procedures in accordance with the methods described above, including
reagents for use in, e.g., nucleic acid amplification procedures (e.g.,
PCR, RT-PCR), DNA sequencing procedures, or DNA labeling procedures. For
example, in certain embodiments, the kit further includes a container
providing a 5' sense primer hybridizable, under primer extension
conditions, to a predetermined polynucleotide template, or a primer pair
comprising the 5' sense primer and a corresponding 3' antisense primer.
In some embodiments, the kit includes one or more containers containing
one or more primers that are fully complementary to single nucleotide
polymorphisms or multiple nucleotide polymorphisms, wherein the primers
are useful for multiplex reactions, as described above. In other,
non-mutually exclusive variations, the kit includes one or more
containers providing nucleoside triphosphates (conventional and/or
unconventional). In specific embodiments, the kit includes
alpha-phosphorothioate dNTPs, dUTP, dITP, and/or labeled dNTPs such as,
e.g., fluorescein- or cyanin-dye family dNTPs. In still other,
non-mutually exclusive embodiments, the kit includes one or more
containers providing a buffer suitable for a primer extension reaction.
In some embodiments, the kit includes one or more labeled or unlabeled
probes. Examples of probes include dual-labeled FRET (fluorescence
resonance energy transfer) probes and molecular beacon probes. In another
embodiment, the kit contains an aptamer, e.g., for hot start PCR assays.

Reaction Mixtures

[0210] In another aspect of the present invention, reaction mixtures are
provided comprising the polymerases with increased 3'-mismatch
discrimination activity, as described herein. The reaction mixtures can
further comprise reagents for use in, e.g., nucleic acid amplification
procedures (e.g., PCR, RT-PCR), DNA sequencing procedures, or DNA
labeling procedures. For example, in certain embodiments, the reaction
mixtures comprise a buffer suitable for a primer extension reaction. The
reaction mixtures can also contain a template nucleic acid (DNA and/or
RNA), one or more primer or probe polynucleotides, nucleoside
triphosphates (including, e.g., deoxyribonucleotides, ribonucleotides,
labeled nucleotides, unconventional nucleotides), salts (e.g., Mn2+,
Mg2+), and labels (e.g., fluorophores). In some embodiments, the
reaction mixture further comprises double stranded DNA binding dyes, such
as SYBR green, or double stranded DNA intercalating dyes, such as
ethidium bromide. In some embodiments, the reaction mixtures contain a
5'-sense primer hybridizable, under primer extension conditions, to a
predetermined polynucleotide template, or a primer pair comprising the
5'-sense primer and a corresponding 3' antisense primer. In certain
embodiments, the reaction mixture further comprises a fluorogenic FRET
hydrolysis probe for detection of amplified template nucleic acids, for
example a Taqman® probe. In some embodiments, the reaction mixture
contains two or more primers that are fully complementary to single
nucleotide polymorphisms or multiple nucleotide polymorphisms. In some
embodiments, the reaction mixtures contain alpha-phosphorothioate dNTPs,
dUTP, dITP, and/or labeled dNTPs such as, e.g., fluorescein- or
cyanin-dye family dNTPs.

EXAMPLES

[0211] The following examples are offered to illustrate, but not to limit
the claimed invention.

[0212] Mutations in Z05 D580G polymerase were identified that provide a
reduced ability to extend an oligonucleotide primer with a 3'-mismatch to
a template. In brief, the steps in this screening process included
library generation, expression and partial purification of the mutant
enzymes, screening of the enzymes for the desired property, DNA
sequencing, clonal purification, and further characterization of selected
candidate mutants. Each of these steps is described further below.

[0213] Clonal Library generation: A nucleic acid encoding the polymerase
domain of Z05 D580G DNA polymerase was subjected to error-prone
(mutagenic) PCR between Blp I and Bgl II restriction sites of a plasmid
including this nucleic acid sequence. The amplified sequence is provided
as SEQ ID NO:33. The primers used for this are given below:

PCR was performed using a range of Mg2+ concentrations from 1.8-3.6
mM, in order to generate libraries with a range of mutation rates. Buffer
conditions were 50 mM Bicine pH 8.2, 115 mM KOAc, 8% w/v glycerol, and
0.2 mM each dNTPs. A GeneAmp® AccuRT Hot Start PCR enzyme was used at
0.15 U/μL. Starting with 5×105 copies of linearized Z05
D580G plasmid DNA per reaction volume of 50 μL, reactions were
denatured using a temperature of 94° C. for 60 seconds, then 30
cycles of amplification were performed, using a denaturation temperature
of 94° C. for 15 seconds, an annealing temperature of 60°
C. for 15 seconds, an extension temperature of 72° C. for 120
seconds, and followed by a final extension at a temperature of 72°
C. for 5 minutes.

[0214] The resulting amplicon was purified with a QIAquick PCR
Purification Kit (Qiagen, Inc., Valencia, Calif., USA) and cut with Blp I
and Bgl II, and then re-purified with a QIAquick PCR Purification Kit. A
Z05 D580G vector plasmid was prepared by cutting with the same two
restriction enzymes and treating with alkaline phosphatase, recombinant
(RAS, cat# 03359123001) and purified with a QIAquick PCR Purification
Kit. The cut vector and the mutated insert were mixed at a 1:3 ratio and
treated with T4 DNA ligase for 5 minutes at room temperature (NEB Quick
Ligation® Kit). The ligations were purified with a QIAquick PCR
Purification Kit and transformed into an E. coli host strain by
electroporation.

[0215] Aliquots of the expressed cultures were plated on
ampicillin-selective medium in order to determine the number of unique
transformants in each transformation. Transformations were stored at
-70° C. to -80° C. in the presence of glycerol as a
cryo-protectant.

[0216] Each library was then spread on large format ampicillin-selective
agar plates. Individual colonies were transferred to 384-well plates
containing 2×Luria broth with ampicillin and 10% w/v glycerol using
an automated colony picker (QPix2, Genetix Ltd). These plates were
incubated overnight at 30° C. to allow the cultures to grow and
then stored at -70° C. to -80° C. The glycerol added to the
2×Luria broth was low enough to permit culture growth and yet high
enough to provide cryo-protection. Several thousand colonies at several
mutagenesis (Mg2+) levels were prepared in this way for later use.

[0217] Extract library preparation Part 1--Fermentation: From the clonal
libraries described above, a corresponding library of partially purified
extracts suitable for screening purposes was prepared. The first step of
this process was to make small-scale expression cultures of each clone.
These cultures were grown in 96-well format; therefore there were 4
expression culture plates for each 384-well library plate. 0.5 μL was
transferred from each well of the clonal library plate to a well of a 96
well seed plate, containing 150 μL of Medium A (see Table 3 below).
This seed plate was shaken overnight at 1150 rpm at 30° C., in an
iEMS plate incubator/shaker (ThermoElectron). These seed cultures were
then used to inoculate the same medium, this time inoculating 20 μL
into 250 μL Medium A in large format 96 well plates (Nunc #267334).
These plates were incubated overnight at 37° C. with shaking. The
expression plasmid contained transcriptional control elements, which
allow for expression at 37° C. but not at 30° C. After
overnight incubation, the cultures expressed the clone protein at
typically 1-10% of total cell protein. The cells from these cultures were
harvested by centrifugation. These cells were either frozen (-20°
C.) or processed immediately, as described below.

[0218] Extract library preparation Part 2--Extraction: Cell pellets from
the fermentation step were resuspended in 25 μL Lysis buffer (Table 3
below) and transferred to 384-well thetinocycler plates and sealed. Note
that the buffer contained lysozyme to assist in cell lysis, and DNase to
remove DNA from the extract. To lyse the cells the plates were incubated
at 37° C. for 15 minutes, frozen overnight at -20° C., and
incubated again at 37° C. for 15 minutes. Ammonium sulfate was
added (1.5 μL of a 2 M solution) and the plates incubated at
75° C. for 15 minutes in order to precipitate and inactivate
contaminating proteins, including the exogenously added nucleases. The
plates were centrifuged at 3000×g for 15 minutes at 4° C.
and the supernatants transferred to a fresh 384-well thermocycler plate.
These extract plates were frozen at -20° C. for later use in
screens. Each well contained about 0.5-3 μM of the mutant library
polymerase enzyme.

[0219] Screening extract libraries for reduced 3' primer mismatch
extension rate: The extract library was screened by comparing the
extension rate of a primer perfectly matched to an oligonucleotide
template vs. the extension rate of a primer with a 3' G:T mismatch.

[0220] The enzyme extracts above were diluted 10-fold for primer extension
reactions by combining 2.5 μl extract with 22.5 μl of a buffer
containing 20 mM Tris-HCl, pH 8, 100 mM KCl, 0.1 mM EDTA, and 0.2%
Tween-20 in a 384-well thermocycler plate, covering and heating for 10
minutes at 90° C. Control reactions with perfect match primer
combined 0.5 μl of the diluted extract with 15 μl master mix in
384-well PCR plates. Extension of the primed template was monitored every
10 seconds in a modified kinetic thermal cycler using a CCD camera (see,
Watson, supra). Master mix contained 50 nM primed primer template, 25 mM
Tricine, pH 8.3, 100 mM KOAc, 0.6×SYBR Green I, 200 μM each
dNTP, 100 nM Aptamer, and 2.5 mM Magnesium Acetate. In order to
distinguish extension-derived fluorescence from background fluorescence,
parallel wells were included in the experiment in which primer strand
extension was prevented by leaving out the nucleotides from the reaction
master mix. Reactions with the 3'-mismatched primer were performed as
above except 1.5 ul the diluted extract was added to each reaction and
1.5 mM Manganese Acetate was substituted for the Magnesium Acetate.
Increasing the amount of extract three fold and using Manganese as the
metal activator both make mismatch extension more likely and therefore
improve the selectivity of the screen for those enzymes with the greatest
ability to discriminate against 3'-mismatch extension.

[0221] Approximately 5000 mutant extracts were screened using the above
protocol. Approximately 7% of the original pool was chosen for
rescreening based on a perfect match primer extension value above an
arbitrary cutoff and low mismatch to perfect match extension ratio.
Culture wells corresponding to the top extracts were sampled to fresh
growth medium and re-grown to produce a new culture plates containing the
best mutants, as well as a number of parental cultures to be used for
comparison. These culture plates were then used to make fresh extracts
which were rescreened to confirm the original screen phenotype. The
primer extension rates for the reactions with the perfect 3'-matched and
the 3'-mismatched primers were calculated as the slope of the rise in
fluorescence over time for the linear portion of the curve. The ratio of
mismatched extension slope divided by the perfect matched extension slope
was used to rank and select the best candidates. Selected clones from the
rescreening, plus for comparison the parental clone Z05 D580G, with their
respective genotypes and phenotypes are included in the table below.

Various Amino Acid Substitutions at the Z05 E493 position: The effect of
various substitutions at the E493 position of Z05 DNA polymerase on
mismatch discrimination in allele-specific PCR was examined. These
substitutions were created in both Z05 DNA polymerase and Z05 D580G DNA
polymerase by PCR-based site-directed mutagenesis or by cloning synthetic
gene fragments into vectors for one or both enzymes and the expressed
mutant enzymes were purified and quantified. Z05 E493 mutants G
(Glycine), K (Lysine), and R (Arginine); and Z05 D580G mutants A
(Alanine), G (Glycine), K (Lysine), and R (Arginine) were compared to
their respective parental enzyme in an allele-specific PCR assay.

[0222] The control DNA polymerases of this example are a Thermus sp. Z05
DNA polymerase of SEQ ID NO:1 or a Thermus sp. Z05 DNA polymerase of SEQ
ID NO:1 except that the amino acid at position 580 is Glycine (e.g., a
D580G substitution) (hereinafter Z05 D580G polymerase).

[0223] Primers were used that amplify a region of the human BRAF gene and
are perfectly matched to the target when said target carries a mutation
in codon 600 of BRAF, V600K. Against wild-type BRAF target, present in
human genomic DNA, the allele selective primer results in a single A:C
mismatch at the 3' end. The common primer is perfectly matched to the
BRAF gene, as is the probe sequence, which allows for real-time, TaqMan
detection of amplification. Each reaction had 10,000 copies (33 ng) of
wild-type Human Genomic cell line DNA, or either 10,000 or 100 copies of
a linearized plasmid containing the BRAF V600R mutant sequence in a final
volume of 16 μl. To allow for the different salt optima of the
enzymes, amplifications were performed using a range of KCl
concentrations from 25 to 130 mM. Buffer conditions were 50 mM Tris-HCl
pH 8.0, 2.5 mM MgCl2, 0.2 mM each dNTP, 0.02 U/μl UNG, and 200 nM
Aptamer. Forward and Reverse primers were at 100 nM and the probe was at
25 nM. All DNA polymerases were assayed at 20 nM and add 2% (v/v) enzyme
storage buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM
EDTA, 1 mM DTT, 0.5% Tween 20) to the reactions. The reactions were
performed in a Roche LightCycler 480 thermal cycler and denatured using a
temperature of 95° C. for 60 seconds, then 99 cycles of
amplification were performed, using a denaturation temperature of
92° C. for 10 seconds and an annealing temperature of 62°
C. for 30 seconds.

[0224] Reactions were in duplicate, crossing points ("Cps") were
calculated by the Abs Quant/2nd derivative Max method and the Cps
were averaged. PCR efficiency was calculated from the slope of the 100
and 10,000 copy perfect match plasmid reactions at the KCl concentration
which resulted in the earliest 10,000 copy perfect match plasmid Cp. High
Copy delta Cp is equal to the difference between the Cp values of the
reactions with 10,000 copy of 3'-mismatched wild-type genomic target and
the Cp values of the reactions with 10,000 copy of perfect match plasmid
target.

[0225] The table below contains the averaged Cp values at the KCl
concentration for each enzyme which resulted in the earliest high copy
plasmid Cp and the calculated PCR efficiency and high copy delta Cp. The
data indicate that several amino acid substitutions at position E493 of
Z05 DNA polymerase result in improved discrimination of primer mismatches
in allele-selective PCR.

[0226] This example demonstrates that the E493A, E493G, E493K, and E493R
mutant enzymes have improved rare allele detection relative to both
control polymerases, Z05 and Z05 D580G.

Example 2

Taq DNA Polymerase with E491K Mutation

[0227] This example shows that a mutation at the E491 position of Taq DNA
polymerase results in increased 3'-mismatch discrimination.

[0228] The homologous amino acid position to E493 in Z05 DNA polymerase is
E491 in Taq DNA polymerase. A synthetic DNA fragment containing an E491K
(Lysine) mutation was cloned into Taq DNA polymerase and the expressed
protein was purified and quantified. Taq E491K DNA polymerase was then
compared to the parental Taq DNA polymerase in an allele-selective PCR
assay.

[0229] The control DNA polymerase of this example is a Thermus sp. Taq DNA
polymerase of SEQ ID NO:2.

[0230] Primers were used that amplify a region of the human BRAF gene and
are perfectly matched to the target when said target carries a mutation
in codon 600 of BRAF, V600K. Against wild-type BRAF target, present in
human genomic DNA, the allele selective primer results in a single A:C
mismatch at the 3' end. The common primer is perfectly matched to the
BRAF gene, as is the probe sequence, which allows for real-time, TaqMan
detection of amplification. Each reaction had 10,000 copies (33 ng) of
wild-type Human Genomic cell line DNA, or either 10,000 or 100 copies of
a linearized plasmid containing the BRAF V600R mutant sequence in a final
volume of 16 μl. To allow for the different salt optima of the
enzymes, amplifications were performed using a range of KCl
concentrations from 25 to 130 mM. Buffer conditions were 50 mM Tris-HCl
pH 8.0, 2.5 mM MgCl2, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.4 mM
dUTP, 0.2 μM aptamer, and 0.02 U/μl UNG. Forward and Reverse
primers were at 100 nM and the probe was at 25 nM. All DNA polymerases
were assayed at 20 nM and add 2% (v/v) enzyme storage buffer (50% v/v
glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5%
Tween 20) to the reactions. The reactions were performed in a Roche
LightCycler 480 thermal cycler and denatured using a temperature of
95° C. for 60 seconds, then 99 cycles of amplification were
performed, using a denaturation temperature of 92° C. for 10
seconds and an annealing temperature of 62° C. for 30 seconds.

[0231] Reactions were in duplicate, crossing points ("Cps") were
calculated by the Abs Quant/2nd derivative Max method and the Cps
were averaged. PCR efficiency was calculated from the slope of the 100
and 10,000 copy perfect match plasmid reactions at the KCl concentration
which resulted in the earliest 10,000 copy perfect match plasmid Cp. High
Copy delta Cp is equal to the difference between the Cp values of the
reactions with 10,000 copy of 3'-mismatched wild-type genomic target and
the Cp values of the reactions with 10,000 copy of perfect match plasmid
target.

[0232] Table 6 shows the averaged Cp values at the KCl concentration for
each enzyme which resulted in the earliest high copy plasmid Cp and the
calculated PCR efficiency and high copy delta Cp. The data indicate that
an E491K amino acid substitution in Taq DNA polymerase results in
improved discrimination of primer mismatches in allele-selective PCR.

[0234] This example shows that polymerases having a mutation at position
5488 of a Thermus sp. Z05 DNA polymerase have increased 3'-mismatch
discrimination.

[0235] The control DNA polymerases of this example are a Thermus sp. Z05
DNA polymerase of SEQ ID NO:1 and a Thermus sp. Z05 DNA polymerase of SEQ
ID NO:1 except that the amino acid at position 580 is glycine (e.g., a
D580G substitution) (hereinafter Z05 D580G polymerase).

[0236] Reactions were in duplicate, crossing points ("Cps") were
calculated by the Abs Quant/2nd derivative Max method and the Cps
were averaged. PCR efficiency was calculated from the slope of the 100
and 10,000 copy perfect match plasmid reactions at the KCl concentration
which resulted in the earliest 10,000 copy perfect match plasmid Cp. High
Copy delta Cp is equal to the difference between the Cp values of the
reactions with 10,000 copy of 3'-mismatched wild-type genomic target and
the Cp values of the reactions with 10,000 copy of perfect match plasmid
target.

[0237] Reaction conditions were as described in Example 1. Table 7 shows
the averaged Cp values at the KCl concentration for each enzyme which
resulted in the earliest high copy plasmid Cp and the calculated PCR
efficiency and high copy delta Cp. The data in Table 7 indicate that
several amino acid substitutions at position 5488 of Z05 DNA polymerase
result in improved discrimination of primer mismatches in
allele-selective PCR.

[0239] This example shows that polymerases having various substitutions at
position F497 of a Thermus sp. Z05 DNA polymerase have increased
3'-mismatch discrimination. These substitutions were created in both Z05
DNA polymerase and Z05 D580G DNA polymerase by cloning synthetic gene
fragments into vectors for one or both enzymes and the expressed mutant
enzymes were purified and quantified. Z05 F497 mutants A (Alanine), G
(Glycine), S (Serine), T (Threonine), and Y (Tyrosine); and Z05 D580G
mutants D (Aspartic Acid), K (Lysine) and S (Serine) were compared to
their respective parental enzyme in an allele-specific PCR assay.

[0240] The control DNA polymerases of this example are a Thermus sp. Z05
DNA polymerase of SEQ ID NO:1 or a Thermus sp. Z05 DNA polymerase of SEQ
ID NO:1 except that the amino acid at position 580 is glycine (e.g., a
D580G substitution) (hereinafter Z05 D580G polymerase).

[0241] Reaction conditions were as described in Example 1. Table 8 shows
the averaged Cp values at the KCl concentration for each enzyme which
resulted in the earliest high copy plasmid Cp and the calculated PCR
efficiency and high copy delta Cp. The data indicate that several amino
acid substitutions at position F497 of Z05 DNA polymerase result in
improved discrimination of primer mismatches in allele-selective PCR.

[0243] It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications or
changes in light thereof will be suggested to persons skilled in the art
and are to be included within the spirit and purview of this application
and scope of the appended claims. All publications, sequence accession
numbers, patents, and patent applications cited herein are hereby
incorporated by reference in their entirety for all purposes.